Regenerative Strategies in Treatment of Peripheral Nerve Injuries in Different Animal Models

BACKGROUND: Peripheral nerve damage mainly resulted from traumatic or infectious causes; the main signs of a damaged nerve are the loss of sensory and/or motor functions. The injured nerve has limited regenerative capacity and is recovered by the body itself, the recovery process depends on the severity of damage to the nerve, nowadays the use of stem cells is one of the new and advanced methods for treatment of these problems. METHOD: Following our review, data are collected from different databases "Google scholar, Springer, Elsevier, Egyptian Knowledge Bank, and PubMed" using different keywords such as Peripheral nerve damage, Radial Nerve, Sciatic Nerve, Animals, Nerve regeneration, and Stem cell to investigate the different methods taken in consideration for regeneration of PNI. RESULT: This review contains tables illustrating all forms and types of regenerative medicine used in treatment of peripheral nerve injuries (PNI) including different types of stem cells " adipose-derived stem cells, bone marrow stem cells, Human umbilical cord stem cells, embryonic stem cells" and their effect on re-constitution and functional recovery of the damaged nerve which evaluated by physical, histological, Immuno-histochemical, biochemical evaluation, and the review illuminated the best regenerative strategies help in rapid peripheral nerve regeneration in different animal models included horse, dog, cat, sheep, monkey, pig, mice and rat. CONCLUSION: Old surgical attempts such as neurorrhaphy, autogenic nerve transplantation, and Schwann cell implantation have a limited power of recovery in cases of large nerve defects. Stem cell therapy including mesenchymal stromal cells has a high potential differentiation capacity to renew and form a new nerve and also restore its function.


Introduction
Traumatic affections of peripheral nerves were the most common serious problems leading to a long-lasting disability including sensory and motor dysfunction, neuropathic pain, muscle atrophy, and even limitation of life [1]. Sciatic, peroneal, tibia, brachial plexus, and radial nerves were the most commonly affected peripheral nerves [2].
The radial nerve is the largest nerve of the brachial plexus supplying all extensors of carpal and digit and even sensation of dorsal region of carpus and digit, its injury occurs as a result of drag, transaction, pressurization, vehicle accident that leads to a complete humeral fracture and also local injections of some drugs [3].
Sciatic nerve is the thickest nerve in the whole body and it is considered the main direct continuation of lumbosacral plexus, descending as distal as the heel of the foot, supplies several muscles in the leg and even sensation of most skin of the lower leg [4][5][6]. Sciatic injuries were commonly caused by compression, stretching or traction, laceration, crushing, and also pelvic fracture [1].
Central nervous system (CNS) has no tendency to heal, while peripheral nerve repair is limited, and complete recovery doesn't occur in most critical conditions. Repairing mechanism is a complex of several pathological interactions such as neuronal and axonal regeneration, Wallerian degeneration, production of inflammatory cytokines, and neurotrophic factors from Schwan cells [7].
Traditionally, Peripheral nerve injuries (PNI) have been managed surgically either by end-to-end or end-to-site anastomosis. Nerve auto-grafting in long nerve defects was considered the gold standard for management of PNI but has some restrictions for obtaining donor nerve, donornerve infection, and neuroma formation [4]. Schwan cell auto transplantation has two major disadvantages including a long time for in vitro growth and culture and also the additional damage to donors [8].
Besides the disadvantages of the previous traditional methods, they don't achieve full nerve recovery and a satisfactory result has not been obtained. Nowadays, medicine is directed to tissue engineering to solve most regardless cases of nerve dysfunction, organ failure, and end-stage disease [7]. Transplantation of allogenic Schwan cells together with a nerve scaffold helps in provoking therapeutic nerve repair and become an applicable method in human cases [4].
Mesenchymal stem cells (MSCS) are multi-potent, plastic, un-differentiated cells that can be obtained from several body sources such as bone marrow, adipose tissue, amniotic membrane, dental pulp, and umbilical cord [9]. MSCS transplantation has proved a successful progress in repairing most damaged tissues and this capacity came from their self-renewal, fast-proliferation, and multi-potent differentiation [8]. Bone marrow mesenchymal stem cells (BMSCS) promote nerve repair through synthesis of neurotrophic factors such as'' nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NTF3), and glial cell-derived neurotrophic factor (GDNF) ''. The BMSCS isolation is considered an invasive technique with a low number of harvested cells to enhance proliferation and differentiation [8]. So, trials are directed toward adipose-derived stem cells which have the same phenotype and genotype regeneration power as BMSCS but rejoice by other features as it can be easily obtained from any fat-rich source, its low immunogenicity, and its faster proliferation than BMSCS [10].
Nerve guidance conduits are tubular structures that can be used for bridging and regeneration of nerve gap defects when injected with extracellular matrix proteins (ECM), growth factors, or supporting cells when autografting is limited due to donor availability [11][12][13]. There were different kinds of artificial nerve conduits such as chitosan, collagen, and poly (DL-lactide-e-caprolactone) and they are considered the most commonly and frequently used in research studies [14,15].
The properties of the ideal nerve conduits were good biodegradability and biocompatibility within the tissue, better porosity and permeability for drug release, and low immunogenicity and toxicity for the body. These characteristics facilitate neural regeneration and stimulate axonal remyelination at the site of injury [16,17]. In our review, we demonstrate various nerve conduits used for the repair of nerve injury and the potential effect of each type in peripheral nerve regeneration.
ADSCs are pluripotent adult stem cells, obtained from any fat-rich sources that had the properties of rapid proliferation and multi-lineage differentiation into osteoblasts, chondrocytes, and adipocytes as BMSCs, but it differs in their higher intensity in fatty tissue after isolation, so it takes a shorter period for tissue regeneration than BMSCs. ADSCs have low immunogenicity power and are rarely rejected by the recipients [8,18,19].
BMSCs are another multipotent adult mesenchymal stem cells that can be easily obtained through aspiration of bone marrow. They can differentiate into bone, cartilage, and fat cells and present neural or glial markers typical of Schwann cells in several neurodegenerative diseases. However, they have low proliferation power than embryonic stem cells and a more invasive method than ADSCs as they require anesthesia in addition to their low cell count after isolation regarding other types of stem cells [20][21][22].
EPCs (Endothelial progenitor cells or umbilical cord mesenchymal stem cells) are proliferative cells produced from fetal tissue after birth with no invasive procedure to the donor or patient and are also available in cell banks. They can be easily and ethically obtained compared with embryonic or bone marrow stem cells, so they are usually preferred over other types. The proliferation capacity and differentiation power of umbilical cord mesenchymal stem cells are proved to be higher when compared with bone marrow mesenchymal stem cells [18,23,24].

Material and method
Our review collected from different databases focusing on research investigating the different treatment strategies of peripheral nerve injuries including therapeutic medicines, nerve conduits, and different types of stem cells in different animal models (rat, mice, rabbit, cat, dog, monkey, pig and sheep hoping that these trials can be used as an ''off-shelf'' medicine in human and animal application.

Peripheral nerve injury (PNI)
Stretch-related injuries are the most common and frequent type of PNI, and their etiology is usually inherited where the nerve nature is very elastic due to its collagenous endoneurium. The nerve injury occurs when it is exposed to a force exceeding its strength and when this force is high enough may result in a full disruption of nerve continuity as in brachial plexus avulsion. Stretch-related injuries occur in the case of nerves that are anatomically related to bones as radial nerve paralysis in complete humeral fracture [25].
The second common type is laceration injuries, which represent about 30% of serious cases, whereas a complete cutting of the nerve or parts from it occurs. This type is mostly involved in research because it is easier to be performed [26].
The third type of PNI is nerve compression which may be induced either by mechanical deformation or ischemia. The most severe form of this type is related to complete disruption of the action potential without any tearing or transection of nerve fibers. So, researchers confused about the pathophysiology of this type either is due to external pressure applied on the nerve or due to induced ischemia especially since there is no, or little histological evidence reported in this type. ''Saturday Night palsy'' due to radial nerve compression was documented and ultra-structural studies showed myelin and axoplasm dis-positioning and nerve fibers degeneration at the compression site more severe than in areas away from the compression [27].

Nerve injury classification and grading
Seddon divided nerve injuries by severity into three broad categories: neurapraxia, axonotmesis, and neurotmesis. Neurapraxia is the simplest form of injury with a mild interruption in nerve impulse conduction, but the continuity of the nerve is not affected. So, it is a transient condition with varying recovery periods. Axonotmesis means discontinuity in axon and myelin sheath with damaged endoneurium and perineurium. Neurotmesis involves disconnection of a nerve with loss of nerve function and recovery without surgical intervention does not usually occur because of scar formation and the loss of the mesenchymal guide that properly directs axonal regrowth [28].
Sunderland's classification system further re-classifies three injury types described by Seddon into five categories depending on the severity of the damage as shown in Table 1 A first-degree injury is equivalent to Seddon's neurapraxia, and a second-degree injury is equivalent to axonotmesis. Third-degree nerve injuries occur when there is disruption of the axon (axonotmesis with partial loss in endoneurium). Seddon's neurotmesis represents fourthand fifth-degree injuries in Sunderland's classification. In a fourth-degree injury, all parts of the nerve are damaged except the epineurium. Five-degree injury is the most severe form in which there is complete dis-connection of the nerve [29].

Neuropathology and mechanism of injury
The peripheral nerve trunk is mainly composed of several nerve fascicles and is surrounded by its connective tissue sheath known as epineurium. Each fascicle consists of groupings of nerve axons entrapping within the endoneurium sheath while the nerve fascicles are surrounded by a different type of sheath called perineurium. The orientation of fibers in each sheath differs from each other, the perineurium and epineurium are circular but the endoneurium is longitudinally aligned. Many blood capillaries are distributed within the epineural sheath and give collateral minor vessels to supply the endoneurial sheath through the perineurium. This vascularization system can aid in introducing a secondary injury to the nerve when expose to severe compression leading to vascular edema causing additional pressure on the nerve structure and contribute to nerve trauma [30]. There are various mechanisms for applying pressure on the nerve and succeeding in nerve injury. Compression of vascular capillaries supplying peripheral nerve when the nerve runs through a narrow anatomical position causes ischemia to the nerve and is categorized as grade 1 injuries or neuropraxia. Traumatic compression by a blunt object as surgical forceps or clamps to an extended period without nerve cutting is usually categorized as a crush injury. Laceration by a knife, gunshot, or glass piece led to the discontinuation of the nerve known as neurotmesis [31].
Sciatic and radial nerve damage may be occurred after intra-muscular injection due to the harmful effect of the injected drug, or the physical trauma caused by in correct method of injection by a less experienced person resulting in sever trouble shock in sensation and may extend to a motor dysfunction [32].

Neural response to injury
A series of degenerative cascades occur after nerve injury which is considered a direct precursor to regeneration. The extent of regeneration is primarily influenced by the degree of initial damage and subsequent degenerative changes. In first-degree, there is only a conduction block and no true degeneration or regeneration; pathological changes are mild or nonexistent. In the second degree, a calcium-mediated process known as Wallerian (or anterograde) degeneration occurs distal to the injury site with little histological change at the injury site or nearby [33].
In third-degree injuries (intra fascicular injuries), a significant trauma-induced local reaction displaces. The elastic endoneurium retracts the ends of severed nerve fiber. Hemorrhage and edema from local vascular trauma cause a strong inflammatory response. The injured segment develops a dense fibrous scar as fibroblasts multiply, which results in fusiform swelling (neuroma) and additional perineural scar tissue [25].

Treatment strategies involved in PNI
In recent years, most candidates are directed to advanced alternative medicines in manipulation of PNI cases instead of previous old surgical interventions like end-to-end anastomosis, donor nerve transplantation, allografting of various types of nerve grafts like vein allograft, and implantation of Schwann cell in the injury site. The recent treatment strategies included different types of stem cells and nerve conduits.

Adipose-derived stem cells (ADSCs)
The ADSCs can be easily harvested from any fat tissue source with no harmful effect and resulted in a high number of cells with very fast culturing and harvesting techniques. It has a good restoration of functional assessment of nerve physiology [34].

Bone marrow stem cells (BMSCs)
Mesenchymal stem cells (MSCs), in particular BMSCs, have been demonstrated to be the greatest candidate for regenerating neural tissues. They could rapidly transform into axons and Schwann cells with little immunogenicity and effective immune regulation. They are extremely proliferative and can change into multiple tissue lineages [35].

Umbilical cord stem cells (Endothelial progenitor cells)
Schwann cells, brain cells, and axons can all be produced by umbilical cord stem cells (hematopoietic stem cells). Their results are quite encouraging, and positive outcomes have been noted but it needs tissue banks [36].

Nerve conduits
A nerve guidance conduit (artificial nerve conduit or artificial nerve graft) is an artificial means of guiding axonal regrowth to facilitate nerve regeneration and it's one of several clinical treatments for nerve injuries. Examples: Collagen nerve conduit, silicone tube, chitosan/fibroinbased nerve scaffold, polycaprolactone nerve conduit (PCL), Silk fibroin -based nerve graft, and polyglycolic acid (PGA) conduit. Using stem cells alone without a guiding material as a nerve graft conduit does not aid in bridging the nerve to re-construct again so most recent research are used these innovative alternatives as a promising guide for cell transplantation [37].

Current progress strategies
Recently, miscellaneous types of stem cells and growth factors have been used to produce efficient results in a limited time either alone or with different types of conduits together. Olfactory ensheathing cells (OECs), Autologous dermal fibroblasts, neural stem cells (NCS), Gingival derived mesenchymal stem cells (GMSCs), basic fibroblast growth factor (bFGF), glial cell line-derived neurotrophic factor (GDNF) and gene transfer of adenoviral bone morphogenetic proteins (AdBMP7) have been conducted in recent studies [18,[38][39][40][41][42] (Table 2).

Animal models of PNI regeneration
Most of the candidates in research had resorted to using animal models to help in the modification of the ideal method of treatment to be applied in human therapy strategies in many urgent cases. Rodents are less similar to human immune system, while dogs, cats, swine, sheep, and non-human primates are considered the most volunteers in resembling the human body physiology and ideal to be used to obtain the best evaluation of functional recovery [62].
Concerning experimental studies using rodents as an animal model, we investigate 58 research demonstrate different treatment strategies like using adipose stem cells, bone marrow stem cells, neural crest stem cells, and other types of mesenchymal stem cells also application of various kinds of nerve conduit as ANA conduit, NeuraWrap TM , fibrin gel conduit and also administration of many drugs help in improving nerve recovery as alfa-lipoic acid, curcumin, Zofenopril, dexamethasone and methyl cobalamin (MeCbl). Induction of injury is either done by crushing using surgical clump or forceps, or by a transaction of a piece of nerve and filling the gap formed in between the two stumps by conduit material. The follow-up period is differed according to many factors like weight, age, species, and also the methods of evaluation used are confined to behavioral analysis as a sciatic functional index, electrophysiological parameters, histopathological analysis, fluorogold retro-tracing assessment, electron microscope, and gene expression by real-time PCR, Figs. 1, 2 and 3 summarized the different regenerative strategies and animal models used in PNI.
In canine studies, different types of mesenchymal stem cells were used to heal the most affected nerves such as radial in the forelimb and sciatic in the hindlimb. Other methods as autografting of nerve after removal, application of nerve guidance conduit, and injection of nerve growth factors and extra-cellular vesicles are also used. Clinical evaluations were conducted by using histopathology, immunohistochemistry, and functional assessment either motor or sensory. The result was obtained by comparison to control group animals in each parameter and many studies were compared to autografting as considered the ideal technical approach in nerve quality healing.
In feline studies, only two research had reported in the peripheral nerve injury model and approach to treatment by grafting normal body tissue like venules or a synthetic sponge material had proved that nerve recovery after implantation was better unless untreated.
In pigs, rabbits, monkeys, and sheep, most of the research was found helpful in manipulation of nerve injury by using stem cell therapy together with nerve grafting after several types of nerve injuries. So, nowadays after all of these articles, there is no difficult way to apply any of these modern approaches when facing different types of nerve injuries by backing and following up the newest published articles.

Effect of animal models in the direction of research study in PNI
Regarding the animal that can be selected according to the effect of different therapeutic methods on an injured nerve, each animal has its point of selection. Murine species (rats and mice) are the most frequently used on a small animal scale, they are considered more economic, easily handled, housed, and investigated in large groups that became more accurate and representative, although, the gap applied in this species (almost not exceed 10 mm) which is relatively shorter than those of a human gap, also suspected time for complete healing is very short compared to human nerve injuries. ultrasonography required for following up the improvement cascade was almost difficult to be applied besides the genomic material of this species being lower than that of human beings [63,64]. Canine and feline species are considered heavily weighted animals. Their weight helped in typical clinical signs of peripheral nerve injuries such as knuckling syndrome in sciatic neuropathy, so facilitate follow-up of the case and the degree of improvement. In addition, this species is relatively more similar to human genomic basis and produces more representative outcomes after a certain period of treatment. On the other hand, this species has an ethical problem with its use in research studies, they require high cost for purchase, housing, and care during the experimental period and the availability is limited in certain countries [65,66].
Rabbits are one of the most commonly used animal models in nerve regeneration studies and their gaps are near to those that occur in humans, but they have a high cost of purchasing and housing, also they need an intense care system. Rabbits' life is short, they rarely exceed five months, so this period is not enough for large gap studies.large animal models like pigs, sheep, equine, and nonhuman primates are limited in experimental studies due to their very high-cost, difficult housing, care, and, evaluation of the study, However, the absolute similarity between human and non-human primates became a promising  challenge for testing the appropriate methods and efficacy of treatment strategies before applied in human being. [67][68][69].
The choice of an appropriate animal model in future research studies depends on the direction of research goals and ideas. Rats and mice will remain the most commonly used models in research studies for their advantages mentioned above, However, human injury defects can be representative by large models such as dogs and monkeys that are limited as their high-cost issues and ethical concerns. So, according to the aims of the designed study and the outcome expected to be obtained, the authors are directed to their best choice of the animal model.

Some clinical trials reported in pni using stem cell therapy
Although several research studies investigate the stem cell protocol in the treatment of animal models undergoing peripheral nerve damage, however in most vet clinic cases using these technologies remains almost rare. But there were various cases documented in human using these trials. A study was conducted by the University of Miami, Florida, United States, they used autologous human Schwan cells augmentation in several nerve injuries (ClinicalTrials.gov Identifier: NCT05541250). Another study was presented by Zhang Peixun, Department of Orthopedics and Trauma, Peking University People's Hospital, they investigate the Mid-term clinical effect of biodegradable conduit small gap tubulation to repair peripheral nerve injury in multi-center (ClinicalTrials.gov Identifier: NCT03359330).
Tissue Eng Regen Med (2023) 20 (6):839-877 867 1-Beginning at 4 weeks, the SFI values in treated groups were higher than the control group ''p\0.05'' 2-The numbers of NF200-positive cells and S100positive cells were significantly larger than in the conduit group. 3-The CMAPs and MNCV values were higher in the treated groups than control (p\0.05) [148]  was present in mid-graft sections from allografts and untreated animals and low no. in distal nerves than in autograft groups [150] TBARS Recently stem cells were used with growth factors such as PRP, glial cell line-derived neurotrophic factor, and basic fibroblast growth factor, more over medicine nowadays was directed to the use of extracellular vesicles of stem cells (exosomes) that help in providing a favorable microenvironment for peripheral nerve regeneration via mediating axonal growth and regulate inflammatory cascade after injury. Overall, we believed that stem cell therapy became an accessible routine in the treatment strategy of different kinds of PNI and is considered the better regenerative medicine for the improvement of traditional old therapeutic interventions.

Conclusion
Peripheral nerve injuries had a limited regeneration capacity particularly when the nerve gap exceeds the possible degree of extend, the nerve axon couldn't be able to reconstruct a new nerve tissue so maintenance of the Peripheral nerve's proper functionality after injury becomes an intractable challenge for clinical researchers. Nowadays, most of the clinical trials involved in treatment of this PNI are directed to different types of mesenchymal stem cell therapies as an alternative to nerve neurorrhaphy to obtain high-quality healed nerve tissue. In our review, we conclude that nerve grafting applications together with stem cells alone or with other growth factors have the best scoring of nerve repairing capacity. After that, the use of nerve conduit alone came later to help Schwan cell in building of new axons and enhance the reverse of Wellerian degeneration. However, other techniques like systemic injection of therapeutic medicine such as nerve tonics and natural antioxidants gave good results in much research and opened a way to interpose in this innovated medicine (Tables 3-8).
Acknowledgement Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). The authors' research incorporated in this article did not obtain any particular grant from funding organizations in the public, or not-for-profit sectors.
Author contribution E-BSH and AAI created the idea for the article. E-BSH, AAI, KMM, and IAM designed the research work, collected the review data, and revised the manuscript draft. E-SMA designed the charts included in the review and all authors reviewed and approved the last version of the manuscript.
Data availability All data collected or analyzed during this study are included in this published review.

Declarations
Conflict of interest There are no conflicts of interest to declare.
Ethical statement There are no animals experiments carried out for this article.
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