Chapter 4 Methods and Protocols in Peripheral Nerve Regeneration Experimental Research: Part I—Experimental Models

doi:10.1016/S0074-7742(09)87004-9

International Review of Neurobiology

Volume 87, 2009, Pages 47-79

https://doi.org/10.1016/S0074-7742(09)87004-9 Get rights and content

This paper addresses several basic issues that are important for the experimental model design to investigate peripheral nerve regeneration. First, the importance of carrying out adequate preliminary in vitro investigation is emphasized in light of the ethical issues and with particular emphasis on the concept of the Three Rs (Replacement, Reduction, and Refinement) for limiting in vivo animal studies. Second, the various options for the selection of the animal species for nerve regeneration research are reviewed. Third, the two main experimental paradigms of nerve lesion (axonotmesis vs. neurotmesis followed by microsurgical reconstruction) are critically outlined and compared. Fourth, the various nerve models that have most commonly been employed are overviewed focusing in particular on forearm mixed nerves and on behavioural tests for assessing their function: the ulnar test and the grasping test which is useful for assessing both median and radial nerves in the rat. Finally, the importance of considering the influence of various factors and diseases which could interfere with the nerve regeneration process is emphasized in the perspective of a wider adoption of experimental models which more closely mimic the environmental and clinical conditions found in patients.

Introduction

Adequate methodological adoption should be the basis for reaching any scientific goal, but unfortunately this requirement isn't always met in nerve regeneration research (Geuna and Varejão, 2008, Geuna et al., 2004, Vleggeert‐Lankamp, 2007). One of the reasons might be the difficulty in obtaining the important methodological information from published research papers, the methods sections of which are usually very synthetic, due to page limit constraints, and often incomplete.

This review is the first of a series of four methodology‐oriented papers that have been included in this special issue on nerve regeneration of the International Review of Neurobiology with the aim of providing the reader with an up‐to‐date critical overview on the important elements that should be considered for designing and carrying out a successful study. While this first paper will address the selection of the experimental models and the study design, the other three reviews will focus on techniques for evaluating study results, namely morphological (Raimondo et al., 2009, this issue), electrophysiological (Navarro and Udina, 2009, this issue), and behavioural (Costa et al., 2009, this issue).

Section snippets

In Vitro Models of Axonal Elongation

The attention given to ethical issues in biomedical research involving animals has greatly increased over the last years. One of the most important achievements is the progressive spread among the scientific community of the “Three Rs” (replacement, reduction, and refinement of animal studies) concept put forward by Russell and Burch (1992).

As far as the first principle, replacement, is concerned, the selection of in vitro models of axon elongation should always be considered for nerve

In Vivo Animal Models for the Study of Nerve Repair and Regeneration

When an investigator wants to move from an in vitro to an in vivo experimental model, it is important to choose the animal model which best fits with the study goals, while taking into consideration the pros and cons of the different options available.

While in most biomedical application rats and mice are by far the two most employed laboratory animals, in nerve regeneration studies there is a clear prevalence of rat use. A PubMed analysis of a random sample of 1500 research papers on nerve

Experimental Lesion Paradigms for Nerve Regeneration Research

Two main experimental lesion paradigms can be adopted for nerve regeneration studies: (1) axonotmesis, that is, induction of nerve fiber interruption by crush injury without discontinuing the nerve; (2) neurotmesis, that is, complete transection of the whole nerve, followed by microsurgical nerve reconstruction. The two lesion paradigms are strictly related to the corresponding clinical conditions observable in patients (Table III) with the only difference that, unlike man, in the rat the crush

Selection of the Nerve Model

The animal body contains many nerves and, although the structure of peripheral nerves is similar (Geuna et al., 2009, this issue), several factors can guide the choice of the nerve model for an experimental study. The main factor is certainly the size of the nerve and, in fact, the large size appears to be the main reason why the sciatic nerve is the most frequently used nerve model (Varejao et al., 2004). In addition, the presence and number of collateral branches should also be considered

Interfering Conditions and Disease Models

Usually, experimental nerve regeneration studies are carried out on ideal subjects, that is, young and healthy animals. This might represent a problem when researchers seek to translate experimental results to the clinics since patients often do not match these two characteristics (i.e., they are not young and/or concurring diseases are present). Since this discrepancy is likely to represent one of the causes of the failure in translating laboratory bench results to the patient bed, the

Conclusions

This methodology‐oriented paper is expected to provide some elements which might facilitate researchers in choosing the best experimental model for their nerve regeneration research. Although we wish to emphasize that there is no absolutely best experimental protocol and thus the choice should be left to each researcher after accurate consideration of many different factors, we would also like to try to put forward some personal recommendations that, of course, do not claim to be the last word

Acknowledgments

This work was supported by grants from the MUR (Italian Ministry of University and Research), ex‐60% fund, FIRB fund (code: RBAU01BJ95), PRIN2005 fund (code: 2005057088), the Compagnia di San Paolo (Bando Programma Neuroscienze), and the Regione Piemonte (Progetto Ricerca Sanitaria Finalizzata).

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Recovery of sciatic nerve with complete transection in rats treated with leuprolide acetate: A gonadotropin-releasing hormone agonist
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In crushed models, the injured axons have the capacity of regeneration and reinnervation in a spontaneous way. The injury level generated in crushed models is axonotmesis, but it is difficult to know the level of axonotmesis, because this depends of the pressure and force administered [20,21]. Nevertheless, in our study we report the effect of long-term LA treatment in rats with sciatic nerve complete transection.
It has been reported that the Gonadotropin-releasing hormone (GnRH) and its agonist leuprolide acetate (LA) can act as promoters of nerve regeneration. The aim of this study is to evaluate the effect of LA in a complete transection model. Sciatic nerve injury (SNI) was performed using a complete nerve transection and immediately repaired by epineural sutures. Rats were divided into three groups: SHAM, SNI treated with LA (SNI + LA) or saline solution (SNI + SS) for 5 weeks. Sciatic nerve regeneration was evaluated by kinematic gait analyzes, electrophysiological, morphological and biochemical tests. SNI + LA group had a functional recovery in kinematic gait, an increase in ankle angle value and a faster walking speed, compound muscle action potential amplitude, nerve conduction velocity (NCV). Furthermore, the number of myelinated axons and microtubule-associated protein 2 (MAP-2) expression were also higher compared to SS group. In conclusion, LA treatment improves of gait, walking speed, NCV, axons morphometry and MAP-2 expression in rats with sciatic nerve complete transection. These results suggest that LA can be a potential treatment for peripheral nerve injuries.
Use of the CatWalk gait analysis system to assess functional recovery in rodent models of peripheral nerve injury – a systematic review
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The femoral nerve, the other main nerve of the hindlimb, is used to some extent, but not as often as the sciatic nerve model, given its smaller diameter and broader array of methods to evaluate functional recovery in the sciatic nerve model. ( Irintchev, 2011; Tos et al., 2009). Experimental models of forelimb PNI, especially the median nerve model, have undergone a kind of renaissance in the recent past (Stossel et al., 2017; Tuffaha et al., 2018; Ronchi et al., 2018; Gluck et al., 2018; Bao et al., 2018; Casal et al., 2018).
Injuries of the peripheral nervous system are common among the population affecting around 3% of all trauma patients. This high clinical need in the field of peripheral nerve injury and regeneration has been steadily driving experimental and epidemiological research. Thereby, it is crucial to determine the exact degree of recovery of end-organ function. Regeneration after nerve injuries is assessed by a wide variety of techniques and pre-clinical model systems, where rodent models are among the most widely used. However, results from rodents are difficult to translate to human patients in general, and reproducible and comparable assessment of functional recovery is of highest importance. Computerized gait analysis allows comprehensive acquisition of locomotor function. As the animals cross the recording device voluntarily, functional recovery is assessable with a minimum degree of human interference on their behavior. This article aims to give a detailed overview on the existing literature on CatWalk gait analysis in rodent models of peripheral nerve injuries of upper and lower extremities, e.g. axonotmesis, neurotmesis or fibrosis, with special emphasis on differences between models. Researchers interested in assessment of locomotor function in such models will especially benefit from this work as it will provide them with an overview of the various experimental setups and expected outcomes. This work also addresses potential pitfalls and hurdles in order to promote well designed, comparable studies allowing for accelerated development of therapeutic strategies in peripheral repair and regeneration.
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However, mammal models are preferable, assuming a goal of eventual use in humans. Tos et al. (2009) gave an account of multiple animal models used in nerve regeneration studies. While Lewis, Sprague Dawley, and Wistar rat strains are all used in PNI studies, regenerative PNI studies almost exclusively use Lewis rats, reportedly because they exhibit the least-problematic behavioral response to the limb numbness which follows nerve transection.
Informational density and relative accessibility of the peripheral nervous system make it an attractive site for therapeutic intervention. Electrode-based electrophysiological interfaces with peripheral nerves have been under development since the 1960s and, for several applications, have seen widespread clinical implementation. However, many applications require a combination of neural target resolution and stability which has thus far eluded existing peripheral nerve interfaces (PNIs). With the goal of aiding PNI designers in development of devices that meet the demands of next-generation applications, this review seeks to collect and present practical considerations and best practices which emerge from the literature, including both lessons learned during early PNI development and recent ideas. Fundamental and practical principles guiding PNI design are reviewed, followed by an updated and critical account of existing PNI designs and strategies. Finally, a brief survey of in vitro and in vivo PNI characterization methods is presented.
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Chapter 4 Methods and Protocols in Peripheral Nerve Regeneration Experimental Research: Part I—Experimental Models

Introduction

Section snippets

In Vitro Models of Axonal Elongation

In Vivo Animal Models for the Study of Nerve Repair and Regeneration

Experimental Lesion Paradigms for Nerve Regeneration Research

Selection of the Nerve Model

Interfering Conditions and Disease Models

Conclusions

Acknowledgments

Biomaterials

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J. Neurosci. Methods

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J. Neurosci. Methods

J. Neurosci. Methods

Neuroscience

J. Hand Surg.

Int. Rev. Neurobiol.

Prog. Neurobiol.

Exp. Neurol.

Surgery

Brain Res.

Mol. Cell Neurosci.

Exp. Neurol.

Int. Rev. Neurobiol.

J. Hand Surg. (Am.)

Neuroscience

Brain Res.

J. Neurosci. Methods

Exp. Neurol.

Eur. Urol.

Exp. Neurol.

Mol. Ther.

Behav. Brain Res.

J. Neurosci. Methods

Neurosci. Lett.

Exp. Neurol.

Brain Res.

C. R. Biol.

Exp. Neurol.

Neuroscience

J. Neurosci. Methods

Exp. Neurol.

Neurochem. Int.

How age impairs the response of the neuromuscular junction to nerve transection and repair: An experimental study in rats

J. Orthop. Res.

Regeneration through long nerve grafts in the swine model

Microsurgery

Peripheral nerve regeneration through allografts compared with autografts in FK506‐treated monkeys

J. Neurosurg.

Peripheral nerve regeneration is delayed in neuropilin 2‐deficient mice

J. Neurosci. Res.

Standardizing nerve crushes with a non‐serrated clamp

J. Reconstr. Microsurg.

Regeneration and functional recovery in the upper extremity of rats after various types of nerve injuries

J. Peripher. Nerv. Syst.

In vitro assessment of axonal growth using dorsal root ganglia explants in a novel three‐dimensional collagen matrix

Tissue Eng.

Motor and sensory re‐innervation of the lung and heart after re‐anastomosis of the cervical vagus nerve in rats

J. Physiol.

Effects of Schwann cells and donor antigen on long‐nerve allograft regeneration

Microsurgery

Inter‐ and intraobserver reliability of walking‐track analysis used to assess sciatic nerve function in rats

Microsurgery

Strain differences in autotomy in rats undergoing sciatic nerve transection or repair

Ann. Plast. Surg.