Animal models used to study direct peripheral nerve repair: a systematic review

Francisco Javier Vela , Guadalupe Martínez-Chacón Alberto Ballestín José Luis Campos Francisco Miguel Sánchez-Margallo,Elena Abellán

1 Department of Microsurgery, Jesús Usón Minimally Invasive Surgery Centre, Cáceres, Spain 2 Scientific Director, Jesús Usón Minimally Invasive Surgery Centre, Cáceres, Spain

Abstract Objective: Peripheral nerve repair is required after traumatic injury. This common condition represents a major public health problem worldwide. Recovery after nerve repair depends on several factors, including the severity of the injury, the nerve involved, and the surgeon's technical skills. Despite the precise microsurgical repair of nerve lesions, adequate functional recovery is not always achieved and, therefore, the regeneration process and surgical techniques are still being studied. Pre-clinical animal models are essential for this research and, for this reason, the focus of the present systematic review (according to the PRISMA statement) was to analyze the different animal models used in pre-clinical peripheral nerve repair studies.Data sources: Original articles, published in English from 2000 to 2018, were collected using the Web of Science, Scopus, and PubMed databases.Data selection: Only preclinical trials on direct nerve repair were included in this review. The articles were evaluated by the first two authors, in accordance with predefined data fields.Outcome measures: The primary outcomes included functional motor abilities, daily activity and regeneration rate. Secondary outcomes included coaptation technique and animal model.Results: This review yielded 267 articles, of which, after completion of the screening, 49 studies were analyzed. There were 1425 animals in those 49 studies, being rats, mice, guinea pigs, rabbits, cats and dogs the different pre-clinical models. The nerves used were classified into three groups: head and neck (11), forelimb (8) and hindlimb (30). The techniques used to perform the coaptation were: microsuture (46), glue (12),laser (8) and mechanical (2). The follow-up examinations were histology (43), electrophysiological analysis(24) and behavioral observation (22).Conclusion: The most widely used animal model in the study of peripheral nerve repair is the rat. Other animal models are also used but the cost-benefit of the rat model provides several strengths over the others.Suture techniques are currently the first option for nerve repair, but the use of glues, lasers and bioengineering materials is increasing. Hence, further research in this field is required to improve clinical practice.

Key Words: nerve; microsurgery; peripheral nerve; regeneration; repair; reconstruction; direct nerve repair;animal model; coaptation; PRISMA; systematic review

Introduction

Peripheral nerve injury is a common disorder in society,with approximately 1 million patients requiring peripheral nerve surgery worldwide every year (Daly et al., 2012). There are many causes of nerve injury: crush, ischemia, sharp damage, traction, and, lesin this field s commonly, electric shock and vibration (Robinson, 2000, 2004). Road accidents are the primary cause of nerve trauma in the civilian population(Huckhagel et al., 2018a) while gunshots wounds, bombs and other explosive devices are the most common causes of nerve trauma in military conditions (Birch et al., 2012).Indeed, the first attempts at nerve repair were initiated by military surgeons during and after wars.

One-third of peripheral nerve injuries are a result of lacerations by sharp objects or long bone fractures (Siemionow and Brzezicki, 2009), with almost three quarters of all nerve injuries occurring in the upper limbs (Huckhagel et al.,2018b), especially affecting the ulnar nerve (Kouyoumdjian,2006). Although axon regeneration has been studied for more than a century, it is still proving a challenge to obtain good functional results regarding nerve repair. There are many factors affecting nerve repair following reconstruction, such as time between injury and treatment, patient age,severity of injury, extension and type of injury (Dvali and Mackinnon, 2007). Furthermore, the technical skills and strategies used by physicians can also affect the success of regeneration. The introduction of microsurgical techniques for nerve repair in 1964 (Smith, 1964) improved the outcomes of nerve reconstruction. The first approach to repair an injured nerve is the end-to-end (ETE) coaptation, performed using epineurial or perineurial suture techniques. However,the types of nerve repair procedures have been extended with different techniques such as end-to-side (ETS) repairs and nerve transfers. One of the most important aspects that should be taken into account in nerve repair is tension, with tensionless repairs shown to result in better outcomes (Griffin et al., 2014).

Understanding nerve regeneration and physiology is crucial to improve functional recovery after peripheral nerve damage and, therefore, further research is needed. Firstly, in vitro studies are required to assess the toxicity and biocompatibility of different drugs, products and materials, while reducing the use of experimental animals according to the Three Rs (3Rs) principle (Kilkenny et al., 2010). However,these assays need to be followed by in vivo studies to investigate tissue reaction, immune system response, vascularization, mechanical function and other variables (Angius et al., 2012). Experimental animals have long been used for research and the results obtained have undoubtedly improved the quality and efficacy of medicine and health. Rodents,carnivores, lagomorphs, pigs, small ruminants and apes are the most common animals used in neuroscience (Mohanty et al., 2019). An ideal translational animal model must reproduce the specific processes that occur in human peripheral nerve injuries. However, each animal model has its own drawbacks and advantages. The identification of appropriate animal models, and their limitations and benefits, is required to produce pre-clinical scientific evidence prior to the development of human clinical trials (Sanders and Young, 1942).

The quality of reporting in systematic reviews (SRs) is not optimal, and only about 10% of SRs report working from a protocol (Moher et al., 2009). Moreover, SRs may fail if the authors do not report the risk of bias in the included studies— an assessment critical to the SR process. The Preferred Reporting Items for Systematic reviews and Meta-Analyses(PRISMA) statement is a 27 item checklist (Moher et al.,2009) that helps researchers improve their SR. With this checklist, the transparency and accountability of SRs will be improved. Simultaneously, if the protocols are registered it will be possible to reduce the number of reviews addressing the same question.

The main objective of this systematic review was to evaluate the different preclinical studies on direct peripheral nerve repair developed between 2000 and 2018, to assess the advantages and disadvantages of each animal model. As specific objectives, we want to study which animal model is better for the different nerves, what examinations we can perform to check the nerve recovery progress and the different techniques that surgeons can use in order to repair nerve injuries.

Data and Methods

Protocol and registration

This SR was performed according to the PRISMA statement(Moher et al., 2009). Being a SR, approval from an ethical committee was not necessary. The search strategy was performed by the first two authors, using the Web of Science,Scopus, and PubMed databases. This review protocol has no PROSPERO registration number because the outcomes are not directly related to human health, thus it is not eligible for inclusion in the International Prospective Register of Systematic Reviews.

Eligibility criteria

This SR of animal studies was not possible to apply all the PICOS (Population, Interventions, Comparator, outcomes and Study design), required on PRISMA statement, since PRISMA was originally devised for clinical trials.

The types of participants considered for this SR were animal models used in peripheral nerve studies, such as mouse,rat, guinea pig, rabbit, cat and dog.

The type of intervention referred to the different preclinical trials used in order to evaluate the best animal model depending on the technique used (ETE or ETS neurorrhaphy)for the peripheral nerve repair and regeneration.

The type of outcomes were measured in respect to nerve repair and regeneration, good level of muscle reinnervation,improvement of axon fibers number, better quality of the regeneration process, the regenerative processes of peripheral nerves after intervention, and the behavioral evolution of animals.

The types of studies eligible for this review were original preclinical trials evaluating direct peripheral nerve repair techniques. Reviews and meeting abstracts were not included. Studies concerning spinal cord or roots, cadaver, robot,flaps, grafts, cranial nerves, training, teaching and transplantation were also excluded.

Information source and search

Studies were identified by searching three electronic databases: Web of Science, Scopus, and PubMed. These databases were systematically searched for English language papers (published from January 2000 to December 2018)by entering the following keywords and Boolean operators:TS=(nerve* AND microsurg* AND (anastomos* OR direct repair) AND (rat OR mouse OR rabbit OR cat OR guinea pig OR dog OR animal)) in Web of Science; and nerve*AND microsurg* AND (anastomos* OR direct repair) AND(rat OR mouse OR rabbit OR cat OR guinea pig OR dog OR animal) in Scopus and PubMed.

Study selection

The titles and abstracts were evaluated for inclusion or exclusion and, when one could not be discarded, the full text of the article was acquired. The flow diagram (Figure 1) details the progression of studies that were collected or excluded,with reasons, in this SR. After reading all of the papers, the authors discussed them and resolved any disagreements by consensus.

Data collection process

The data collected from the papers was recorded in a table(Table 1) for later analysis. The information extracted from articles was: First author, year of publication, title, and journal (in order to organize them); technique used to perform the neurorrhaphies (suture, glue, laser and mechanical); animal model (number of animals used, groups of study, species, nerve model, anesthesia used and the duration of the study); and follow-up exams (histology, electrophysiological study and behavioral observations). Also, a brief conclusion and comments on each article were noted (data not shown).

Risk of bias in individual studies

In order to ascertain the validity of the eligible studies, thefirst two authors of this SR used the Systematic Review Centre for Laboratory Animal Experimentation's (SYRCLE's) risk of bias tool (Hooijmans et al., 2014) for animal studies. Ten entries related to 6 types of bias are contained in this risk of bias tool, and the types of bias were: selection, performance,detection, attrition, reporting and other bias. For each entry there was a signaling question that had to be answered with:“yes” (low risk of bias), “no” (high risk of bias) or “unclear”(unclear risk of bias). If one of those questions was answered with “no”, indicates a high risk of bias for that entry.

Table 1 Summary of the studies included in this review about peripheral nerve repair

Table 1 Continued

The reviewers worked independently to determine the adequacy of randomization and concealment of allocation,baseline characteristics, blinding of patients and outcomes,randomization of housing and outcomes, incomplete data,and the selective reporting. The qualification of each risk of bias was categorized as low, high or unclear.

Statistical analysis

No statistical analyses were performed due to the big variety of species included in this study.Yan et al., 2002; Isla et al., 2003; Wieken et al., 2003; Beer et al., 2004; Choi et al., 2004; Dourado et al., 2004; Cho et al.,2010) were recorded, which fell to 14 (Hwang et al., 2005,2006, 2008; Isaacs et al., 2005; Liu et al., 2005; Lutz and Lidman, 2005; Ozkan et al., 2005; Peker et al., 2005; Landegren et al., 2006; Tos et al., 2008; Hu et al., 2009; Kokkalis et al.,2009; Kostopoulos et al., 2009; Wang et al., 2009) between 2005 and 2009, 10 (Cho et al., 2010; Nunes e Silva et al.,2010, 2012; Attar et al., 2012; Fox et al., 2012; Omori et al.,2012; Papalia et al., 2012; Félix et al., 2013; Knox et al., 2013;Wu et al., 2013) from 2010-2014, and finally 7 (Bao et al.,2016; Adel et al., 2017; Bhatt et al., 2017a, b; Fekrazad et al.,2017; Hasturk et al., 2018; Liu et al., 2018) published in the most recent period (2015-2018).Animals

Of the 49 studies, 3 (Howard et al., 2000; Isla et al., 2003; Fox et al., 2012) contained some groups examining graft or gap repair, but those groups were not included in this SR.

From all of the studies included, a total of 1425 animals were used: 1226 rats, 101 rabbits, 27 mice, 39 cats, 24 guinea pigs, and 8 dogs.

Interventions

Not all of the studies employed the same nerve model, so we divided the articles into 3 groups: head and neck, forelimb,and hindlimb nerves. The head and neck group comprised 11 studies, using guinea pig (1), cat (1), dog (1), rabbit (2),and rat (6) animal models for the study of facial (6), hypoglossal (3), vagus (1), and recurrent laryngeal (1) nerves. For the forelimb group, we only found 8 studies, with mouse (1)and rat (7) used as animal models to focus on the musculocutaneous (5), median (5), and ulnar (2) nerves. In the hindlimb group, 30 studies were found, using mouse (1), rabbit(3), and rat (26); with the nerves of interest being the sciatic(21), peroneal (6), tibial (4), saphenous (1), and femoral (1).All of this information is documented in Figure 2.

There was high degree of variability in the duration of the studies, ranging from 1 to 96 weeks. Many studies (Lutz et al., 2000; Zhang et al., 2000; Papakonstantinou et al., 2002;Park et al., 2002; Suri et al., 2002; Menovsky and Beek, 2003;Wieken et al., 2003; Dourado et al., 2004; Hwang et al., 2005;Peker et al., 2005; Kostopoulos et al., 2009; Wang et al., 2009;Fox et al., 2012; Wu et al., 2013; Bao et al., 2016; Liu et al.,2018) have more than one end study period, which means that there are in total 89 end time periods out of the 49 selected studies. Similarly to the year of publication analysis,the length of the studies were grouped according to the following periods: 1 to 4 weeks (group 1), 5 to 8 weeks (group 2), 9 to 12 weeks (group 3), 13 to 16 weeks (group 4), 17 to 24 weeks (group 5), and longer than 24 weeks (group 6). The results are shown in Table 2.

The different routes of administration for anesthesia were also analyzed, with the following results: intravenous (1), inhalation (3), subcutaneous (3), intramuscular (6), intraperitoneal (33), and 3 undefined.

One of the main focuses of this SR was on the different

Figure 1 Flow diagram of study selection.

Results

Study selection

As depicted in the PRISMA flow diagram (Figure 1), we collected a total of 401 results from our search in the Web of Science, Scopus, and PubMed databases. After removing duplicates (134), 267 papers remained, which were recorded and screened according to their titles and abstracts. From these 267 articles, we excluded 172 because they were book chapters or reviews, or because their title did not fit with the criteria, then a further 41 that were judged not to accord with the criteria based on their abstracts. The full text of the remaining 54 articles was assessed for eligibility, of which one contained only the abstract in English, with the rest of the paper written in another language, so this paper was discarded. Finally, examination of the full text from the remaining 53 studies identified 3 articles about graft repair and 1 studying the viability of a gel, rather than nerve repair or regeneration. Once the exclusion of articles was complete,we had 49 studies that used small animal models: dog (1),cat (1), guinea pig (1), mouse (2), rabbit (5) and rat (39), and all of these were included in the qualitative synthesis.

Ultimately, 49 original articles about direct nerve repair and published in English were selected for inclusion in this review.

Study characteristics

Methods

First we analyzed the year in which each study was published, in order to assess the activity of research in this field.We classified the articles into groups of five year periods, and the results show a decrease in publication rate. In the first five years (2000-2004), 18 articles (Al-Qattan, 2000; Giovanoli et al., 2000; Howard et al., 2000; Lutz et al., 2000; Zhang et al., 2000; Menovsky and Beek, 2001, 2003; Shamir et al.,2001; Tiangco et al., 2001; Park et al., 2002; Suri et al., 2002;methods to repair an injured nerve and the materials used for that purpose. The anastomoses can be performed by approximating the nerve edges from the epineurium or the perineurium of the fascicles. Epineurial coaptation was the approximation technique used in 41 of the studies, the perineurial technique was performed in 6, a full thickness neurorrhaphy was used in one study and 3 reports did not describe the type of anastomosis used. There are only two ways to directly repair a nerve that has suffered a trauma:ETE or ETS coaptation. The number of studies that performed an ETE or ETS neurorrhaphy was 40 and 14, respectively, because some of them combined or compared both techniques. Four different techniques were used to carry out the nerve coaptation: suture, glue, laser, or mechanical. Of the 49 studies, 46 used microsuture techniques in order to perform the neurorrhaphies, 12 used glue, 8 used laser and 2 used mechanical techniques. There was only one study that did not specify the technique that they used (data not shown). The suture technique is the most common way to repair the nerve, compared to glue, laser, and mechanical methods. The size range of suture used was between 8-0 and 12-0, with 10-0 the most commonly used, and with nylon sutures being the material of choice in 70% of the studies. As previously mentioned, glues were used in 12 studies: 9 with fibrin glue and 3 with cyanoacrylate. The most widely used laser in preclinical trials was CO2, employed in 6 studies,including 1 study which compared the CO2laser with a potassium titanyl phosphate (KTP) laser. One study examined KTP laser effectiveness and one used a diode laser together with a protein solder. Mechanical techniques were reported in only 2 studies, 1 using titanium clips VCS?and the other using a 1.5 mm coupler to perform the nerve coaptation (data not shown).

Figure 2 Types of nerves used in preclinical peripheral nerve repair studies

Table 2 Peripheral nerve repair preclinical studies: postoperative follow-up period, number of studies and animal models

The follow-up exams used in the studies were classified according to the type of exam. Histological analyses were performed in 43 studies and the objective of the evaluation is shown in Figure 3A. Meanwhile, 24 articles reported different electrophysiological examinations (Figure 3B). Finally the other exams that were described in 22 studies were behavioral observations of the animals. Because the target nerve in each study was not the same, these were not consistent for all studies. We therefore organized the behavioral observations according to the nerve model (Table 3).

Outcomes

Both greeted me with that Parisian countenance13 that said: Yes, I speak English, but you ll have to struggle with your French if you want to talk to me

In all studies, the primary outcome was nerve repair and regeneration, measured by axon recovery identified through histology. In addition, some studies explored muscle reinnervation by electromyography, while others recorded the behavioral observations.

Risk of bias within studies

Table 4 shows the SYRCLE's risk of bias tool (Hooijmans et al., 2014), with the information of bias extracted from the studies. The types of bias extracted were: selection bias, performance bias, detection bias, attrition bias and reporting bias. The highest risks were found in the allocation concealment (selection bias) and blinding (performance bias), with only one study that described the method of concealment and provided the information to show that the trial was effectively blind. According to detection bias, there were some studies with the outcome assessor blinded and selected the animals randomly for outcome assessment. There were no studies with high risk of bias regarding the attrition and reporting bias.

Discussion

Summary of evidence

Registration of SRs may reduce the risk of multiple reviews addressing the same question (Liberati et al., 2009) and, consequently, we attempted to register the present SR in PROSPERO, an international prospective register of SRs. However,this SR was not eligible for inclusion because the outcomes of our included studies are not directly related to human health.

There has been a gradual decrease in the number of publications in the field of direct peripheral nerve repair, which may be due to the promotion of new therapeutic techniques,such as autografts, allografts or conduits (Lovati et al., 2018),as alternative methods to reestablish the connection in a nerve gap (Eren et al., 2018).

One of the factors affecting nerve regeneration is the time lapse from damage to intervention. In 2005 and 2013, two studies addressed this issue, demonstrating that there are no notable differences between immediate nerve repair and that performed 2 weeks (Peker et al., 2005) or 4 weeks (Wu et al., 2013) after injury, with the only difference being the amount of tissue that has to be removed from the injured nerve with the passing of time. Suture techniques are the most common method used in order to repair a nerve injury. However, glue and laser techniques are becoming suitable alternatives for performing nerve coaptation (Menovsky and Beek, 2001; Wieken et al., 2003). As such, it is necessary to compare both alternatives with suturing. A number of authors have reported similar outcomes whether using glues or sutures for ETE coaptations (Menovsky and Beek, 2001;Suri et al., 2002; Dourado et al., 2004; Landegren et al.,2006; Attar et al., 2012; Félix et al., 2013; Knox et al., 2013).Yet others promote the use of glue over sutures (Adel et al.,2017), which may be due to the progress of research into glues. In 2004, the use of glues became a meaningful alternative to sutures when nerve injury occurs in confined anatomical locations (Choi et al., 2004). Also, it is possible to perform ETS neurorrhaphy with glue, obtaining no significant difference in outcome compared to suture techniques(Nunes e Silva et al., 2010, 2012). Nevertheless, it is demonstrated that glues in ETS coaptation with epineurial window had better outcomes that without window (Papalia et al.,2016; Geuna et al., 2017). In a comparison between organic and inorganic glues (fibrin and cyanoacrylate, respectively),fibrin is reported to be more suitable, due to its lower induction of foreign body and inflammatory reactions (Wieken et al., 2003). Regarding lasers, the first results were similar to those obtained with sutures, but with limitations (Menovsky and Beek, 2001). With advances in technology, lasers have become a viable alternative for the performance of nerve anastomosis (Hwang et al., 2005, 2006, 2008) and, some studies now recommend lasers over suture techniques (Bhatt et al., 2017a, b; Fekrazad et al., 2017), possibly due to the increased quality of laser technology. Only one study combines the use of suture and laser, using only two stitches of different sutures to approximate the nerve ends (Menovsky and Beek, 2003), and the conclusion of this study was that polyglycolic acid (PGA) stimulates less foreign body reaction than nylon, and is best used together with a laser. Finally, laser use was combined with chitosan, improving the outcomes in nerve repair (Bhatt et al., 2017a).

Figure 3 Postoperative assessment after peripheral nerve reconstruction.

Table 3 Behavioral observations and frequency classified by nerve

In 2002, Park et al. used titanium clips and concluded that this was a faster technique with a statistically comparable outcome compared to suture neurorrhaphy. Meanwhile,in 2005, a coupler was used and reported to be a suitable alternative to sutures (Lutz and Lidman, 2005). The limitations with these approaches are the foreign body reaction,inflammatory response, uncertainty regarding endoneurium damage when using clips, and the size of the coupler. Mechanical techniques, such as clips, have been proposed to be faster rather than suturing, and could provide an alternative approach to reduce operating time when the nerve is not the only tissue affected (Park et al., 2002). However, the coupler has fewer benefits in nerve regeneration compared with suture neurorrhaphy due to its rigidity and prevention of the crisscrossing of regenerating axons (Lutz and Lidman, 2005).

In the other articles, the suture technique was used to perform the coaptations, and the variability between them is extensive due to the size of the suture and the material used. The standard suture is size 10-0 and made of nylon,but alternative sizes and materials may be used. As the rat was the most commonly used animal model, and the size of rat nerves are very small, use of the smallest suture available increases the ease of the procedure. Suture size 12-0 was used in mice for a similar reason. Regarding types of neurorrhaphy, ETS gave worse results than ETE (Lutz et al.,2000; Liu et al., 2005, 2018; Kokkalis et al., 2009; Papalia et al., 2012), but remains a valid option when tension cannotbe avoided while attempting to perform an ETE coaptation(Zhang et al., 2000; Isaacs et al., 2005). In addition, the outcomes of ETS anastomoses can be improved using ancillary treatments. These compounds used in conjunction with nerve coaptations were: anti-adhesion barrier gel (Isla et al.,2003), oral administration of creatine (Ozkan et al., 2005),neuronal nitric oxide synthase (Wang et al., 2009), platelet rich plasma and mesenchymal stem cells (Cho et al., 2010),

and rat or human amniotic membrane (Hasturk et al., 2018),insulin-like growth factor-I (Tiangco et al., 2001), acetyl-L carnitine (Kostopoulos et al., 2009) or irradiation of the spinal cord with an low power laser (Shamir et al., 2001). It is demonstrated that acetyl-L carnitine prevent the sensory neuronal loss after peripheral nerve injury and it has neuroprotective effect (Wilson et al., 2007). There are currently no clear improvements in nerve regeneration using pharmacological methods, but potential candidates for enhancing nerve regeneration could emerge in the foreseeable future(Panagopoulos et al., 2017).

Table 4 Summary of SYRCLE's risk of bias

Rats are the most commonly used animals for preclinical trials of direct peripheral nerve repair. Although there is insufficient evidence to support the use of other species, such as dog, cat, guinea pig or mouse (Tos et al., 2008; Hu et al.,2009; Cho et al., 2010; Attar et al., 2012; Félix et al., 2013),the rabbit may provide a possible alternative to rat (Giovanoli et al., 2000; Park et al., 2002; Beer et al., 2004; Dourado et al., 2004; Hwang et al., 2008), with their larger and thicker nerves. However, the relatively low cost of rats compared to these alternative models presents a significant advantage.Rats have a brachial plexus structure very similar to human beings (Bobkiewicz et al., 2017), the experimental results using rodent forelimb models are more commonly translated to operating theaters (Tos et al., 2008), but it is necessary to take into account that rodents have a faster regenerating capacity compared to humans (Zhang et al., 2000; Wu et al.,2013). On the other hand, rabbits have more active masseter movements, jaw development, and bigger size and weight than rats, thus they are a really good animal model studying head and neck nerves as facial (Hwang et al., 2008). Despite the big number of studies using rats as animal model in hindlimb nerves, it is not the best model to investigate because it translation to human beings has shown to be unreliable for nerve regeneration (Kaplan et al., 2015). However rats should only be employed in questions about basic science, where background data strongly supports a model's validity. Finally, the number of nerve fibers and nerve size of dogs are close to human ones, making this animal model perfect to practice the nerve coaptation in similar conditions than in clinical practice (Attar et al., 2012).

The biggest limitation of the murine models in nerve repair is the length of the nerves and the difficulty to avoid tension during their repair. For these reasons, the rodent may be a poor option when studying nerve regeneration in a gap, conduit or graft model (Félix et al., 2013; Griffin et al.,2014; Kaplan et al., 2015).

Follow-up exams are also important to determine the extent of regeneration. Histology is possibly the most important assessment, but it can only be analyzed once, at the end of the study. In order to evaluate the progression of nerve regeneration, it is beneficial to be able to examine the model at different time points in the same study (Mackinnon et al., 1991). Electrophysiological analysis can reveal whether or not there is nerve recovery while the animal is still alive,but only concerning motor nerves (Kanaya et al., 1996). Behavioral observations yield a lot of information about motor function recovery, but this can be dependent on the nerve of interest. For example, the motor function of median nerve can be study using the grasping test, but walking track analysis is the most used examination in order to determine the sciatic nerve motor function (Papalia et al., 2003). In this context, the sciatic nerve is the gold standard of hindlimb nerves; the facial nerve, for head and neck nerves; and the median and musculocutaneous nerves in the forelimbs (de Medinaceli et al., 1982; Berg and Kleinfeld, 2003).

Limitations

Outcome level

The present SR combines data across studies with the goal of determining suitable animal models for any peripheral nerve studies. The main limitations of this review are the variety of nerve models and the large variability in the length of the studies.

Study and review level

Our work has a number of limitations, including the choice of language, because there may be more articles that could be included according to the inclusion criteria, but written in languages other than English. The use of anastomosis as search word instead of coaptation has to be taken into account, because it is a more appropriate terminology for nerve repair and possibly many studies could be missed in our review.

Conclusions

Between the years 2000 and 2018, the number of publications addressing preclinical trials of direct nerve repair has decreased, implying that researchers have been focusing on other fields of nerve repair. Comparing the different techniques currently available, the suture and glue methods are effective options, because their use results in promising outcomes, but the laser method is still being debated. Depending on the nerve of interest, the animal model may vary, for example rats are more indicated for studying forelimb nerves while rabbits represent a better option for facial nerve. To study nerve recovery, the protocol should include a histological study, an electrophysiological analysis and the observation of behavioral parameters appropriate to the nerve of interest.

Acknowledgments:The systematic review was performed at the Jesús Usón Minimally Invasive Surgery Centre (CCMIJU) which is part of the ICTS “NANBIOSIS.” We appreciate the help of Krisztina Juhos (Semmelweis University, Hungary) at the beginning, for the collaborative study on the possibilities of PRISMA statement when the systematic review was just an idea.

Author contributions:Conceptualization, data curation, formal analysis, investigation, methodology, writing - original draft: FJV. Investigation, methodology, supervision, writing - review & editing: GMC. Conceptualization, supervision, writing - review & editing: AB. Supervision:JLC and FMSM. Supervision, writing - review & editing: EA. All authors approved the final manuscript.

Conflicts of interest:We declare no conflicts of interest related to this manuscript.

Financial support:None.

Reporting statement:This work followed the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) statement.

Biostatistics statement:The statistical analysis of this study was not necessary.

Copyright license agreement:The Copyright License Agreement has been signed by all authors before publication.

Data sharing statement:For data sharing, individual participant data will be available. Also, the study protocol and informed consent form will be made available beginning 3 months and ending 5 years following article publication to investigators whose proposed use of the data has been approved by an independent review committee identified to achieve aims in the approved proposal. In order to gain access, data requestors will need to sign a data access agreement. Proposals should be directed tofjvela@ccmijesususon.com.

Plagiarism check:Checked twice by iThenticate.

Peer review:Externally peer reviewed.

Open access statement:This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-Non-Commercial-ShareAlike 4.0 License, which allows others to remix, tweak,and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.

Open peer reviewer:Michele R Colonna, Universita degli Studi di Messina, Italy.

Additional file:Open peer review report 1.