Regenerative Peripheral Nerve Interfaces (RPNIs) in Animal Models and Their Applications: A Systematic Review

Regenerative Peripheral Nerve Interfaces (RPNIs) encompass neurotized muscle grafts employed for the purpose of amplifying peripheral nerve electrical signaling. The aim of this investigation was to undertake an analysis of the extant literature concerning animal models utilized in the context of RPNIs. A systematic review of the literature of RPNI techniques in animal models was performed in line with the PRISMA statement using the MEDLINE/PubMed and Embase databases from January 1970 to September 2023. Within the compilation of one hundred and four articles employing the RPNI technique, a subset of thirty-five were conducted using animal models across six distinct institutions. The majority (91%) of these studies were performed on murine models, while the remaining (9%) were conducted employing macaque models. The most frequently employed anatomical components in the construction of the RPNIs were the common peroneal nerve and the extensor digitorum longus (EDL) muscle. Through various histological techniques, robust neoangiogenesis and axonal regeneration were evidenced. Functionally, the RPNIs demonstrated the capability to discern, record, and amplify action potentials, a competence that exhibited commendable long-term stability. Different RPNI animal models have been replicated across different studies. Histological, neurophysiological, and functional analyses are summarized to be used in future studies.


Introduction
Regenerative Peripheral Nerve Interfaces (RPNIs) represent a groundbreaking approach at the intersection of biomedical engineering, neurology, and regenerative medicine.These interfaces have the potential to revolutionize the field of bionic prostheses by facilitating communication between the nervous system and external devices and deterring the development of neuromas [1][2][3][4][5][6][7].Unlike traditional neural interfaces, which rely on electrodes implanted into nerves, RPNIs aim to create a more seamless connection by harnessing the regenerative capacity of peripheral nerves [8].
The aim of this study was to perform the first systematic literature review of the RPNI technique across animal models.The different applications and characteristics of each model are analyzed.We believe the knowledge of all of the different surgical techniques and the different histological, neurophysiological, and functional tests may be useful for future research projects involving RPNIs.

Search Strategy
A comprehensive literature review was executed by searching the MEDLINE/PubMed and Embase databases spanning from 1 January 1970, to 30 September 2023.The search process encompassed both automated and manual approaches, ensuring the identification of all pertinent literature.The adherence to the PRISMA statement (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) [60] guided the execution and reporting of this review.Employing English keywords along with Boolean logical operators, specifically "(RPNI) OR (Regenerative Peripheral Nerve Interfaces)", facilitated the search process.Notably, no limitations were imposed during the search.

Selection Criteria (Figure 1)
We included articles written in either English or Spanish that either described or employed the RPNI technique.Exclusion criteria encompassed studies where the RPNI technique was not utilized or was applied in non-animal models.We omitted duplicated studies and articles from the same author or author groups if they were identical.The evaluation of titles, abstracts, and full text, as well as the application of inclusion and exclusion criteria, was carried out independently by two independent plastic surgeons (J.G. and A.A.M.).Full versions of potentially relevant studies were procured for further assessments.Additional articles were considered following a review of the references from the retrieved articles.In cases of disagreement between the two reviewers, resolution was achieved through discussion and consensus.

Data Extraction
The data were gathered using the software "Microsoft Excel for Mac, version 16.8 (23121017)".The collected data encompassed various elements: the article database, the university center where the study took place, the publication year, the study's objectives and categorized groups, the animal species, along with the total animal count used, specifics about the nerve and muscle utilized in constructing the RPNI, the design and model of RPNI construction, the selection of histological parameters (including muscle angiogenesis, tissue viability, muscle axonal regeneration, neuroma formation, and fibrosis/scarring), a subset of functional variables (encompassing stimulus intensity and localization, compound muscle action potential (CMAP), motor unit action potential (MUAP), compound sensory nerve action potential (CSNAP) measurements, latency periods, and maximum muscle force), the outcomes, and the average follow-up duration.
A comprehensive quantitative analysis of the quality and limitations of the selected studies was conducted.This process was carried out following the "10 Essential ARRIVE (Animal Research: Reporting In Vivo Experiments)" outlined in the guidelines of "ARRIVE guidelines 2.0" [61].Each study was rated on a 10-point scale, considering the degree of compliance with the evaluated items.These criteria represent the minimum requirement of information necessary to ensure that reviewers and readers can assess the reliability of the presented findings.

Data Extraction
The data were gathered using the software "Microsoft Excel for Mac, version 16.8 (23121017)".The collected data encompassed various elements: the article database, the university center where the study took place, the publication year, the study's objectives and categorized groups, the animal species, along with the total animal count used, specifics about the nerve and muscle utilized in constructing the RPNI, the design and model of RPNI construction, the selection of histological parameters (including muscle angiogenesis, tissue viability, muscle axonal regeneration, neuroma formation, and fibrosis/scarring), a subset of functional variables (encompassing stimulus intensity and localization, compound muscle action potential (CMAP), motor unit action potential (MUAP), compound sensory nerve action potential (CSNAP) measurements, latency periods, and maximum muscle force), the outcomes, and the average follow-up duration.
A comprehensive quantitative analysis of the quality and limitations of the selected studies was conducted.This process was carried out following the "10 Essential ARRIVE
The University of Michigan emerges as the foremost research institution in the domain of the RPNI technique employed in animal models, with a noteworthy presence in twentyeight (80%) publications.In twenty-four instances, it stands alone as the primary research center, while in four instances, it collaborates with the universities of Alberta, British Columbia, Delaware, and Groningen.The remaining seven (20%) articles originate from the universities of Wuhan, Beijing, Florida, Cambridge, and Texas (Table 1).The average duration of follow-up for the animal subjects spanned 4.7 months (range, 0-20 months).The longest-running research extended for twenty months [62].
With the purpose of assessing the quality of the included research, a comprehensive quantitative analysis of the 35 studies was conducted.The evaluation resulted in a final average score of 8 out of 10, demonstrating a strong level of compliance with minimum reporting standards.
A different type of Burrito RPNI construction has been published [31] using a segment of a free muscle graft wrapping an intact peripheral nerve (the muscle is placed above the epineurium of a nerve, which is not sectioned).

Aim of the Study
We found four main types of studies regarding the research question: 1.
Neuroma formation within the RPNI was evaluated in twelve articles [1,9,13,15,23,24,26,31,38,[46][47][48].Generally, neuroma formation was not evident in the RPNI; however, it has been observed to increase proportionally with an escalation in the muscle graft mass exceeding 300 mg [13].The rate of neuroma formation is higher when employing the targeted muscle reinnervation (TMR) technique as opposed to RPNI and with the Burrito-RPNI in comparison to the Inlay-RPNI [46,48].Analytical techniques encompass direct visual examination through electron microscopy and staining procedures involving hematoxylin-eosin, Toluidine blue, and Masson's trichrome, as well as the use of anti-alpha bungarotoxin and anti-neurofilament 200 (NF200) monoclonal antibodies and ultrasonography.
Fibrosis formation within the RPNI was scrutinized in sixteen articles [1,9,10,13,15,23,24,26,30,31,35,38,40,46,47,62].In general, fibrosis formation is not conspicuous in the RPNI; however, it has been observed to increase proportionally with an augmentation in the muscle graft mass beyond 300 mg [13].Notably, the rate of fibrosis formation is higher when the electrode is positioned intramuscularly, but lower when it is placed epimysially [10].Analytical techniques encompass direct visual examination via electron microscopy and staining techniques employing hematoxylin, eosin, and Masson's trichrome, as well as the utilization of anti-alpha smooth muscle actin (α-SMA) filament monoclonal antibodies.
The array of studies encompasses other investigations, including the quantification of neuronal density, measurement of the apoptosis index via the Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling (TUNEL) method, measurement of marker expression (Bax, BCL-2, and NTs), and the assessment of the degree of autotomy [25,26].
In the case of another common model (Burrito RPNI using EDC and tibial/peroneal nerve in rats) [24,31,48], the average CMAP measured 4.33 mV with a range of 0.75 to 35.3 mV, the average CSNAP was 123.3 µV with a range of 78.6 to 206.6 µV, and the mean latency was 1.175 ms with a range of 0.8 to 1.55 ms.The mean maximum muscle contraction strength was 2478.8 millinewtons (mN) with a range of 2226.7 to 2933.9 mN.The stimulus intensity was not recorded in any of these models.
When considering Rhesus macaques models [3,62,63], the average stimulus intensity in the nerve was 10.5 µA with a range of 1 to 20 µA, and in the muscle, it was 45 µA with a range of 30 to 90 µA.The average CMAP was 500 mV with a range of 400 to 600 mV.The maximum muscle strength was not evaluated in any of these models, and stimulus intensity data were not recorded for these models as well.

Discussion
The University of Michigan's prominent presence in twenty-eight out of the thirty-five reviewed articles underscores its leadership in researching the RPNI technique in animal models.We hope that other institutions will validate and advance RPNI applications in the near future.
Given that performing RPNIs is not technically difficult, we anticipate an increase in the utilization of animal models and RPNI applications for pain management in humans.A longer path is expected in the case of the RPNI and myoelectric prosthesis, as it entails the need for more extensive technical resources, including the prosthetic device itself and the connection between the RPNI and the prosthesis.

Type of Model
The predominance of rat models in thirty-two instances is a common practice due to their widespread use in biomedical research.Rhesus macaques, while representing a different order of magnitude in complexity, were utilized in only three studies [3,62,63].While rats provide practical advantages, such as cost-effectiveness and ease of handling, the translation of findings to larger primates and ultimately to humans may face challenges given the considerable biological differences.
The distinction between Inlay-RPNI and Burrito-RPNI designs provides valuable insights into the diversity of methodologies.We have observed that the Inlay-RPNI design is recently more often used in published articles than the Burrito-RPNI design.A recent study has demonstrated that the Inlay-RPNI model yielded superior outcomes in preventing neuromas compared to the Burrito-RPNI [48].In the near future, we anticipate the identification of the most suitable RPNI model for specific applications.
The preference for the common peroneal nerve and EDL muscle in RPNI construction is consistent across studies.We believe that the nerve and muscle selection is not so critical to reproduce the RPNI model.However, the combination of two nerves from the same extremity (such as peroneal and tibial nerve [10,27,28]) may induce difficulties in carrying out some basic activities, such as walking or feeding.
It is worth noting that the RPNI technique has been assessed in various clinical studies involving humans, showcasing promising outcomes in alleviating neuropathic pain and in the application of myoelectric prostheses.Nevertheless, future researchers should prioritize addressing the dearth of clinical trials that substantiate these findings [12,50,54,57,58,64,65].

Aim of the Study
We have categorized our analysis into four distinct aims based on the research question.This offers a structured approach to understanding the multifaceted aspects of RPNI applications.The identified aims (neuroma prevention, myoelectric prostheses development, histological analysis, and neurophysiological analysis) encompass a broad spectrum of RPNI applications, demonstrating the versatility and potential of this technique.
We think the use of the RPNI model for myoelectric prostheses could be the most promising application.Being able to obtain the information from different peripheral nerve fascicles could be a paradigm change in peripheral nerve surgery.However, we identified only six studies focusing mainly on myoelectric prosthesis development [3,10,11,34,62,63].One of the main limitations of RPNIs is the difficulty of getting the electrical signal from the muscle to the prosthesis.As we have previously summarized, the low amplitude of the electrical signal and the small size of the muscle graft are the main drawbacks.A subcutaneous electromyographic recording could facilitate the acquisition, amplification, and transmission of the electrical signal from the RPNI to the prosthesis.

Histological Analysis
One of the main concerns when analyzing RPNIs is the blood supply of the muscle graft.Muscle tissue is known for its high demanding oxygen requirements [66,67].The combination of these high muscle metabolic rates and the absence of an established vascular system may hinder efficient oxygen delivery, elevating the risk of complications, like necrosis.Muscular neoangiogenesis, tissue viability, axonal regeneration, or neuroma and fibrosis formation has been evaluated in eighteen publications.No necrosis or muscle graft failure was reported in the animal series analyzed in this review.However, the size of the muscle graft has been associated with the above parameters.Muscle grafts mass exceeding 300 mg presented with worse tissue viability and higher rates of complications, such us fibrosis [13].
Vascularized RPNIs (using vascularized muscle, but not a muscle graft) have been reported, and promising results focusing on neuropathic pain have been published [56,[68][69][70].Despite previous studies analyzing the vascularization of muscle grafts in standard RPNIs, we believe that vascularized RPNIs should yield more stable results.Vascularization of the RPNI is one of our primary concerns, particularly when considering the potential use of a needle to obtain an electric signal from the muscle graft.
One notable limitation within the examined studies lies in the deficiency of objectivity in the histological analysis of samples across diverse research investigations.This shortfall is attributed to the absence of standardized criteria that would facilitate the comparison of histological findings across these studies.The inclusion of subjective terms, such as "good", "viable", or "healthy" introduces inherent ambiguity, thereby impeding the ability to conduct comprehensive comparative analyses among the various studies.Addressing this limitation necessitates the establishment of clear and standardized criteria, which is crucial for promoting objectivity and enhancing the reliability of histological assessments in future research endeavors.

Limitations and Future Challenges
We believe that this systematic review will be very useful in aiding future researchers to enhance surgical techniques and the application of RPNIs across various animal models.Given the significant technical complexity involved in using RPNIs for electrical signal acquisition and myoelectric prosthesis control, we are convinced that refining the animal model of RPNIs could directly impact its application in human contexts.This advancement may signify a significant step towards optimizing procedures and the future viability of RPNIs in clinical applications.
The use of RPNI involves substantial challenges in its clinical implementation.Tissue viability (given its nature as non-vascularized muscle grafts) or limitations in detecting and amplifying electrical signals in RPNI directly impact their functional effectiveness.Transitioning RPNI models from an animal to a human setting presents potential obstacles, given the potential influence of physiological variations on their effectiveness and response.Finally, configuring and adapting patients to prostheses derived from RPNI pose challenges in terms of acceptance and optimal functionality in daily life.These aspects underscore the complexity and potential barriers to be addressed during the development and implementation of RPNI and their clinical applications.

Conclusions
To the best of our knowledge, this is the first systematic review of the RPNI technique in animal models.Murine models of RPNIs have consistently demonstrated promising results among several studies, particularly in the myoelectric prosthetics field and the prevention of neuropathic pain.Histological, neurophysiological, and functional analyses are summarized to be used in further studies.Forthcoming research should aim to validate these findings and continue to improve the synergy between humans and machines, advancing a more sophisticated interaction paradigm.

Table 1 .
General data of studies.

Table 2 .
RPNI models and components of studies.

Table 3 .
Histological analysis of studies.

Table 4 .
Neurophysiological analysis of studies.