Evaluation of Postoperative Nerve Regeneration in Murine Experimental Models Subjected to Nerve Transection

1. Introduction

Peripheral nerve injury (PNI) has been garnering increased attention due to its rising prevalence alongside rapid societal developments[1]. The main causes of PNI, such as diabetes mellitus, autoimmune diseases, infections, medications, and traumatic injuries, exhibit diverse clinical manifestations. Literature reviews indicate a prevalence of 8% for non-traumatic cases[2] and 5% for traumatic instances[3]. Despite advancements in microsurgical techniques, patients with PNI still face unsatisfactory recovery prognosis[4].

Injuries are categorized into neuropraxia, axonotmesis, and neurotmesis, each with distinct implications for treatment and functional recovery[5,6].This scenario underscores the need for innovative therapeutic approaches that could offer more promising outcomes.

In this context, adipose-derived stem cells (ADSCs) emerge as a promising source for regenerative therapies due to their ability to differentiate into various cell types, such as Schwann-like cells, and to release regenerative factors through paracrine mechanisms[7,8].

Scientific literature highlights the potential of these cells in treating peripheral nerve injuries in animal models, with evidence of their role in promoting regeneration and improving nerve function. Experimental studies have been primarily conducted in rodent models, as their nerves share similar size, fascicular organization, and morphology to humans[9]. Regarding the use of ADSCs in treatment, the potential to contribute to the recovery of damaged nerves makes them promising candidates for future clinical applications[10].

Additionally, complementary techniques such as the stromal vascular fraction (SVF) and nanofat are explored to optimize the regenerative potential of ADSCs with paracrine and immunomodulatory actions to induce tissue regeneration. These approaches combine the cellular richness of the SVF with the benefits of the extracellular matrix and paracrine signals provided by nanofat, enabling the development of more effective treatments against nerve injuries[11,12].

In this context, this systematic review aims to present the state of the art in the current literature regarding the use of murine models subjected to peripheral nerve injuries. Subsequently, it intends to synthesize the main methods of analyzing nerve regeneration and their respective results in the selected articles.

2. Materials and Methods

2.1. General Information

This is a systematic search study following Cochrane standards, and this manuscript was conducted according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) 2020 guidelines[13], with data research conducted in February 2024. As this is a systematic literature review, this study was exempt from institutional review board approval and did not utilize external funding sources. The databases MEDLINE, Scielo, and LILACS were used to search for articles addressing peripheral nerve regeneration in animals subjected to nerve transection. The following keywords were used in MEDLINE: ("rat"[All Fields] OR "mice"[All Fields] OR "rabbits"[All Fields] OR "macaca"[All Fields] OR "swine"[All Fields] OR "muridae"[All Fields]) AND ("peripheral nerve injuries"[All Fields] OR "denervation"[All Fields]) AND ("microsurgery"[All Fields] OR "neurosurgery"[All Fields] OR "mesenchymal stem cells"[All Fields] OR "adipose tissue"[All Fields]) AND "nerve regeneration"[All Fields]. The same terms were used in Embase, Scielo, and LILACS with necessary modifications according to the rules of these databases.

2.2. Data Selection and Extraction

Following data research, article selection was conducted by three independent reviewers in two stages: analysis of article titles and abstracts, followed by full-text review. Any conflicts were resolved by a third author. The literature was restricted to articles in English, Portuguese, and Spanish without date limitations. Inclusion criteria were limited to articles assessing peripheral nerve regeneration as the primary outcome and considering the section of the peripheral nerve studied. Moreover, only experimental clinical trials were included since they are the primary studies with the highest impact, thus providing more reliability for the data the authors aimed to analyze. Exclusion criteria were defined as follows: articles that did not address the presence of a comparator group among the studied subgroups; those that did not include methods for the functional evaluation of the studied nerve regeneration; and those that did not use adipose derivatives in at least one subgroup. After applying the inclusion and exclusion criteria, 19 studies were included for full-text analysis. It is important to note that the choice of inclusion and exclusion criteria was made a priori.

Regarding data extraction, it was performed by three independent reviewers who collected the following information from the articles: author, publication year, intervention, objective, conclusion, study location, animal follow-up, population, number of animals per subgroup, breed of animal, functional analyses, histological analyses, and other parameters assessing nerve regeneration. Data on funding sources were also collected.

3. Results

The literature search yielded 273 records, mainly from the MEDLINE database. Abstract screening removed the majority of the articles. The main causes for removal were wrong study design and studies that did not use adipose tissue byproducts.

Figure 1. Flowchart representing the literature search.

Figure 1. Flowchart representing the literature search.

Figure 2. Country of the institution to which the first author is affiliated.

Figure 2. Country of the institution to which the first author is affiliated.

Figure 3. Analytic view of publishing countries.

Figure 3. Analytic view of publishing countries.

The country that produced the most results was China (N=4), followed by Switzerland.

Figure 4. Animal species used in the experiments retrieved.

Figure 4. Animal species used in the experiments retrieved.

Despite including several animal species in our search terms, we only located studies conducted in muridae. The most commonly used species was the Sprague Dawley rat, followed by Wistar and Lewis rats. FVB rats and the mouse were used in a single study each. The average number of subjects was 32.5, being the average number of subjects per intervention group 10.16.

Figure 5. Observation time of the subjects.

Figure 5. Observation time of the subjects.

Most articles performed their intervention immediately after neurotmesis, and then observed the rat for a mean number of 11.26 weeks.

Figure 6. Nerve injury models utilized.

Figure 6. Nerve injury models utilized.

The sciatic nerve injury with a gap of 10mm was the overall most commonly utlized model by a far margin (N = 11). Other gaps were utilized in smaller nerves or animals. One study utilized a chronic denervation model by severing the peroneal nerve and suturing the ends away from each other.

Figure 7. Interventions of control groups.

Figure 7. Interventions of control groups.

38.8% of control groups utilized empty conduits. All of the articles utilized a control group in accordance to the intervention.

Figure 8. Materials utilized in the nerve during interventions by number of articles.

Figure 8. Materials utilized in the nerve during interventions by number of articles.

Figure 9. Materials utilized in the nerve during interventions by total number of nerves severed.

Figure 9. Materials utilized in the nerve during interventions by total number of nerves severed.

ASCs with no further processing were the most common intervention, utilized in a total of 14 articles and 122 nerves. 68/4% of the articles utilized conduits to bridge the nerve gaps. Aside from ASCs, other adipose tissue byproducts utilized were SVF and fat grafts.

4. Discussion

Based on current research, tissue bioengineering and microsurgery emerge as some of the fundamental pillars of regenerative medicine. As intervention studies on vital structures progress, the techniques described increasingly approach practical applicability.

4.1. General Aspects

Among the selected studies, China contributed the highest number (n=5), followed by Switzerland (n=3). This find is compatible with the recent advances that China has been making in the academic publishing scene[33]. Remarkably, Chinese articles had the second-highest average journal impact factor, surpassed only by Korea. This indicates that China has had an increase not only in quantity, but in quality of their publications.

4.2. Fat Grafting and Stem Cells

In accordance with our inclusion criteria, all articles involved ASC-based interventions in at least one group, totaling 269 nerves undergoing sectioning and ASC interventions. The use of ASCs has been gaining popularity within the literature since its discovery as a potential material for tissue regeneration[34]. A search on PubMed for 'Adipose-derived stem cells' shows an increasing trend, indicating growing interest in this topic. Several uses of ASCs are currently under investigation, with the main ones including wound healing, bone healing, immunomodulation, and nerve regeneration[35]. ASCs have gained special attention in the field of stem cell research because they are widely accessible throughout the body, easy to process, and lack the ethical controversies associated with other stem cell sources[36].

The first article proposing the use of ASCs for neuronal regeneration was published in 2005, followed by numerous animal and human research models exploring its therapeutic potential[37]. Their applications across various specialties, including ophthalmology (optic nerve regeneration), urology (treatment of nerve-induced erectile dysfunction), and reconstructive plastic surgery, have been extensively studied[38,39,40].

4.3. Trauma Mechanism

Studies on ASCs in nerve regeneration remain predominantly confined to animal models, notably involving the sciatic nerve neurotmesis with a 10mm gap. Among the analyzed articles, 11 studies employed this specific model, while others explored neurotmesis involving different gaps or other nerves, such as the brachial plexus and the facial nerve. Additionally, several studies investigated crush injury models[41].

The selected studies exhibited a broad spectrum of methodologies and approaches, leading to considerable variability in the types of functional and histological analyses performed. This diversity hindered the feasibility of conducting a systematic review, which generally demands a more standardized approach to data synthesis and analysis. Consequently, we adopted a narrative review methodology to provide a comprehensive and interpretive synthesis of the existing literature[42].

4.4. Functional Analysis

In this study, eight articles[16,19,20,21,22,25,26,31] utilized the "Sciatic Functional Index" (SFI) for evaluating nerve function. Based on Medicaneli et al. (1982)[43], the method is reliable for assessing muscle strength index and tissue electrophysiology, thus evaluating the performance of nerve function in the injured lower limb. For instance, the regeneration of the sciatic nerve was assessed by Dai et al. (2013)[19] with recovery rates ranging from 20.4% to 23.4% over a three-month period. Similarly, Orbay et al. (2012)[44] reported a functional recovery rate of 24.9% within two months in mice that received the intervention, demonstrating the method's effectiveness in analyzing this variable[45,46,47,48]. Conversely, other studies also used alternative scales. Mohammadi et al. (2015)[22], in addition to the SFI, employed the "Basso, Beattie, and Bresnahan for limb motor function" (BBB) and "Static Sciatic Index" (SSI) for assessing the same parameter. Although less used in the reviewed articles, both have validation in the scientific literature for their use[49,50]. Only one article used the "Peroneal Functional Index" (PFI), although the literature equally validates its use compared to the SFI[51].

Regarding methods of nerve electrostimulation, the parameter Compound Muscle Action Potential (CMAP) was used in five articles[15,17,18,28,30], Compound Nerve Action Potential (CNAP) in one article[25], and Nerve Conduction Velocity (NCV) in four articles [16,19,20,25].

The CMAP is intended to measure the sum of action potentials of the concerned muscle fibers, focusing on the response of the innervated muscle[52]. Notably, the experiment by Di Summa et al. (2011)[12] assessed nerve regeneration through significant increases in axonal diameter and CMAP using either autograft or fibrin seeded with ASCs on the PNI. The validation of this method is also present in the literature[53].

The use of CNAP also proved to be a satisfactory parameter in the study by He et al. (2016)[25], being effective in comparing the proposed regeneration in the Lv-ADSCS group versus the 34a-ADSCS group, which proved more effective in the latter.

The measurement of nerve conduction velocity is also an important factor for evaluating innervation of the site, and depends on factors such as axon diameter, myelin sheath thickness, and the length of the "internodes"[54,55]. According to Raisi et al. (2014)[20], NCV was effective in ascertaining poorer performance in nerve regeneration in the pro-inflammatory MSC group, with a lower conduction velocity rate (72%). This aspect was crucial in questioning which factors were triggering, such as: smaller diameter of regenerating axons, thinner myelin sheaths, or even an immaturity of myelinated nerve fibers in that group.

Regarding the Muscle Weight Ratio (MWR), nine articles used this method[17,19,21,22,23,25,26,29,31]. This parameter appears to be used due to the ease of its application in studies, often being the only type of functional evaluation performed[23,29]. In the experimental trial conducted by Pollari et al. (2018)[56], the method proved effective in comparing nerve regeneration in the transection versus crushing of the peripheral nerve group.

4.5. Histological Analysis

The use of optical microscopy alongside software for image analysis was highlighted in all selected articles. Based on Kappos et al. (2015)[21], this combined method was crucial for measuring axonal diameter and fiber diameter. Thus, the thickness of myelin and other important average parameters for evaluating local tissue regeneration can be calculated and compared across groups[57].

The light microscope method was enhanced by complementary analyses in several studies. Notably, the use of toluidine blue in five articles[22,23,25,27,29] emphasized its role in staining myelinated structures that facilitate neuronal visualization[58,59,60]. According to Ozkan et al. (2016)[27], this method proved effective for verifying regular myelinated pre-neuronal bodies with increased diameter in the study group, demonstrating better organization of nerve tissue regeneration.

The scientific literature indicates the efficiency of immunofluorescence methods for analyzing the regeneration of Schwann cells and peripheral remyelination[61,62]. In agreement, Kastamoni et al. (2023)[32] found that the expression of EphA4 was associated with a negative effect on the axonal repair of the sciatic nerve, consistent with previous studies [63,64,65].

Despite its requirement for considerable time and specialized training, transmission electron microscopy is widely mentioned in the literature as a method for obtaining detailed, high-quality images of nervous structures[66,67,68,69]. In this regard, several studies have used this method to obtain highly specific images of structures[16,18,20,25,28,29,30].

4.6. Other Analysis

Tremp et al. (2015)[24] identified a correlation between axonal length measured by MRI and axonal length measured by immunohistochemistry, reaffirming the nerve damage between the sciatic nerve of the intervention versus control. Thus, it is understood that the use of this method can add comparative value between the analyses performed.

Flow cytometry is an effective tool in studies of axonal nerve regeneration, due to its ability to quantitatively analyze individual cells in large populations[70,71,72]. One study used this method to evaluate specific cellular groups and their tissue behavior in regeneration[29].

RNL is a prospective method for evaluating nerve regeneration that compares neuronal expansion through its connections[73,74,75]. Di Summa et al. (2011)[15], evaluated using this method and electrostimulation that the autograft group and the ASCs group showed greater motoneuron regeneration compared to the control, which is in dialogue with current literature[76,77,78].

4.7. Strengths and Limitations

This review fulfills its role of assessing the current state of the scientific literature regarding the use of murine models for the experimentation of peripheral nerve regeneration, as well as elucidating and analyzing the main functional assessment models used in such experimental trials. Another point analyzed was the global distribution of scientific production for this article model, with an emphasis on comparing the use of different fat derivatives, such as ADSCs, SVF, and NanoFat. However, although it has a well-defined study population and outcomes, the breadth of types of peripheral nerve injury makes the execution of a meta-analysis unfeasible, due to the heterogeneity of the selected articles concerning the type of intervention proposed in each study. Lastly, the casuistry of the subgroups is not equivalent, which complicates a more complex biostatistical analysis.

5. Conclusion

The techniques and procedures described in this review hold significant potential for future practical application in peripheral nerve regeneration, as further studies continue to advance the field. Thus, this work serves its purpose by elucidating the current evidence on the topic, evaluating the principal models and formats of experimental studies that permeate the literature. Additionally, it enhances the clarity of the main types of fat derivatives and the methods for evaluating nerve regeneration in the most methodologically rigorous articles reviewed.