International Journal of Molecular Sciences Article Repair of Long Peripheral Nerve Defects in Sheep: A Translational Model for Nerve Regeneration Estefanía Contreras 1,2 , Sara Traserra 1,2 , Sara Bolívar 1,3 , Joaquím Forés 1,4 , Eduard Jose-Cunilleras 5 , Ignacio Delgado-Martínez 1,3 , Félix García 5 , Esther Udina 1,3 and Xavier Navarro 1,3, * 1 Institute of Neurosciences, Department Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain 2 Integral Service for Laboratory Animals (SIAL), Faculty of Veterinary, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain 3 Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas (CIBERNED), 28031 Madrid, Spain 4 Hand and Peripheral Nerve Unit, Hospital Clínic i Provincial, Universitat de Barcelona, 08036 Barcelona, Spain 5 Department of Animal Medicine and Surgery, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain * Correspondence: xavier.navarro@uab.cat; Tel.: +34-93-5811966 Abstract: Despite advances in microsurgery, full functional recovery of severe peripheral nerve injuries is not commonly attained. The sheep appears as a good preclinical model since it presents nerves with similar characteristics to humans. In this study, we induced 5 or 7 cm resection in the peroneal nerve and repaired with an autograft. Functional evaluation was performed monthly. Electromyographic and ultrasound tests were performed at 6.5 and 9 months postoperation (mpo). No significant differences were found between groups with respect to functional tests, although slow improvements were seen from 5 mpo. Electrophysiological tests showed compound muscle action potentials (CMAP) of small amplitude at 6.5 mpo that increased at 9 mpo, although they were significantly lower than the contralateral side. Ultrasound tests showed significantly reduced size of tibialis anterior (TA) muscle at 6.5 mpo and partially recovered size at 9 mpo. Histological evaluation Citation: Contreras, E.; Traserra, S.; of the grafts showed good axonal regeneration in all except one sheep from autograft 7 cm (AG7) Bolívar, S.; Forés, J.; Jose-Cunilleras, group, while distal to the graft there was a higher number of axons than in control nerves. The results E.; Delgado-Martínez, I.; García, F.; indicate that sheep nerve repair is a useful model for investigating long-gap peripheral nerve injuries. Udina, E.; Navarro, X. Repair of Long Peripheral Nerve Defects in Sheep: A Keywords: large animal model; long gap; nerve injury; nerve regeneration; sheep Translational Model for Nerve Regeneration. Int. J. Mol. Sci. 2023, 24, 1333. https://doi.org/10.3390/ ijms24021333 1. Introduction Academic Editor: Tessa Gordon Peripheral nerve injuries result in partial or total loss of motor, sensory, and au- Received: 7 November 2022 tonomic functions of the affected nerve territory [1]. When the axon is transected, the Revised: 29 December 2022 segment distal to the lesion disconnects from the neuronal body and undergoes Wallerian Accepted: 7 January 2023 degeneration [1–4] to create a permissive environment for regeneration. In parallel, the ax- Published: 10 January 2023 otomized neuron switches to a pro-regenerative state, with phenotypic changes to support axon re-growth [5]. Despite adult peripheral neurons having this intrinsic regenerative capability and being able to reinnervate their target organs eventually, functional outcome is not always complete [1,6,7], leading to chronic functional impairments and decreased Copyright: © 2023 by the authors. patient quality of life. Licensee MDPI, Basel, Switzerland. The severity of the nerve injury is one of the main factors that determines the degree of This article is an open access article recovery. Therefore, mild injuries, such as compression or crush, in which only the axons are distributed under the terms and transected but all the connective layers, including the endoneurial tubules, are preserved, conditions of the Creative Commons usually recover normal function. In contrast, when both axons and connective tissue are Attribution (CC BY) license (https:// transected, functional outcomes are worse. After a complete nerve transection, surgical creativecommons.org/licenses/by/ 4.0/). repair is mandatory to suture the proximal and distal nerve stumps together and, thus, Int. J. Mol. Sci. 2023, 24, 1333. https://doi.org/10.3390/ijms24021333 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023, 24, 1333 2 of 17 facilitate that axons can cross the gap and grow along the distal stump. When direct suture of the two stumps is not possible because the separation is too large, a bridge is required to guarantee continuity between the two nerve stumps. Currently, the gold standard for this type of lesion is the interposition of a segment of another nerve from the same patient, called an autologous graft [6,8]. However, long nerve defects cannot always be repaired with an autograft due to the limited availability of nerve sources for the repair of extensive or multiple nerve injuries. In addition, autograft harvesting can lead to neuroma formation and permanent loss of sensation in the territory of the donor nerve [9]. Thus, regeneration of long nerve defects remains a challenge in the clinic, and further research is needed to find alternatives to autograft repair. Tube repair, the implantation of a tube or conduit from natural or synthetic biomaterials to bridge a nerve gap, emerged as a potential alternative to the autograft repair of transected peripheral nerves [10]. However, depending on the size of the injured nerve and the species, the regeneration across nerve conduits is limited by the length of the gap. Experimental studies showed that regenerating axons can bridge empty tubes made of silicone or synthetic materials along a gap of up to 4 mm in the mouse [11], up to 10 mm in the rat [10], and 30 mm in primates [12,13], but fail in most cases with longer gaps. Therefore, the most relevant clinical need for alternative repair methods in human patients are lesions resulting in gaps between 3–30 cm in length [14]. The rat sciatic nerve transection and repair model is the most widely used rodent model in peripheral nerve regeneration [15–19]. Nerve anatomy of rodents has been well studied, but regeneration is faster than in humans and, obviously, only short nerve gaps can be studied [17,20,21]. In addition, the recovery of limb function may be better because of the short regeneration distances between the injured nerve and the target organs in rodents [22]. It is fundamental to provide good experimental models with the aim to explore and design new and innovative therapies with translational potential. Large animal models allow for longer gaps and longer regeneration distances that mimic the clinical conditions often found in human nerves. The development of new clinical approaches in peripheral nerve regeneration includes preclinical animal testing in animal models that reproduce the regeneration process that occurs in human nerve injuries [17]. There is no standard large animal model for nerve repair studies [9]. The large animals used for peripheral nerve injury and regeneration studies include primates, dogs, cats, pigs, and sheep [17,23–25]. The sheep has gained interest as one of the most relevant animal models for preclinical studies [25–28]. Compared to other large animals, sheep have advantages because of their availability, simplicity of care and housing, cost, and social acceptance [20,29,30]. They have generated interest for the study of long nerve gap repair because their peripheral nerves resemble human nerves in terms of their length, diameter, and function [9,20,31]. Protocols for nerve surgery and the clinical evaluation of deficits and histological processing have been recently proposed [25]. The aim of this study is to standardize a model of peripheral nerve injury in sheep and adequate methods for evaluation during a long follow-up period that will be useful for the investigation of new therapeutic alternatives to the autograft for the repair of severe, long-gap nerve injuries. We include the surgical approach, functional monitoring, electrophysiological and ultrasound tests, and histological analyses, for a comprehensive and detailed quantification of nerve regeneration and reinnervation. 2. Results 2.1. Clinical Observations The surgical approach allowed the dissection of the peroneal nerve over a long distance, its resection, and its repair by interposing the same nerve segment with either a 5 or a 7 cm autograft, AG5 and AG7, respectively. All the sheep recovered well from the surgery and survived to the end of the study. In addition, no significant clinical signs were observed during the experimental study. After the surgery, animals were able to stand and walk and showed good mobility. As a result of the peroneal nerve injury, two animals from AG7 group had a marked foot drop posture and developed pressure ulcers. A molded plastic The surgical approach allowed the dissection of the peroneal nerve over a long dis- tance, its resection, and its repair by interposing the same nerve segment with either a 5 or a 7 cm autograft, AG5 and AG7, respectively. All the sheep recovered well from the Int. J. Mol. Sci. 2023, 24, 1333 3 of 17 surgery and survived to the end of the study. In addition, no significant clinical signs were observed during the experimental study. After the surgery, animals were able to stand and walk and showed good mobility. As a result of the peroneal nerve injury, two animals from AG7 splint was placed at group postoperation 1 month had a marked foot drop (mpo), posture and andulcers the skin developed werepressure ulcers. A treated with chlorexhydine and Blastoestimulin cream and covered with a Vetrap bandage. After onewere molded plastic splint was placed at 1 month postoperation (mpo), and the skin ulcers month, one oftreated withrecovered the sheep chlorexhydine and and Blastoestimulin the splint cream In was removed. and coveredthe contrast, with a Vetrap other sheep band- age. After one month, one of the sheep recovered and the splint was removed. In contrast, continued with the foot drop posture until the end of the study. the other sheep continued with the foot drop posture until the end of the study. 2.2. Functional Evaluation 2.2. Functional Evaluation After the surgery, all sheep showed deficit in locomotion score based on the occurrence After the surgery, all sheep showed deficit in locomotion score based on the occur- of foot drop during rence offast walking. foot drop duringIn the resting fast orthostatic walking. position, In the resting all the sheep, orthostatic position,except all thethe sheep, two animals of AG7 group indicated above, were able to maintain the except the two animals of AG7 group indicated above, were able to maintain theplantar support of the plantar right hindlimb. In allofthe support thesheep, we observed right hindlimb. In allevidence the sheep,ofwe foot drop during observed evidencefast of walking, as foot drop during they failed tofast maintain walking,theasplantar support they failed in some to maintain thesteps plantar(scored support asin−some 1 or − 2) (Figure steps (scored1A). as −1 or The proprioceptive response −2) (Figure 1A). The was not significantly proprioceptive responsereduced was not after the surgery significantly and after reduced did not the sur- change during gery theand did not (Figure follow-up change during 1B). The themuscle follow-upmass(Figure of the1B). Thereinnervated right muscle mass tibialis of the right anterior (TA) reinnervated muscle showed tibialis anterior a clear (TA) muscle reduction showed one month aftera clear reduction the surgery in one month after comparison to the surgery in comparison to the contralateral muscle. A significant the contralateral muscle. A significant improvement (p < 0.01) was detected in AG5 group improvement (p < 0.01) at the end of the follow-up with respect to values at 30 days (Figure 1C). The withdrawaldays was detected in AG5 group at the end of the follow-up with respect to values at 30 (Figure 1C). The withdrawal reflex response to pinching the skin of the dorsum of the foot reflex response to pinching the skin of the dorsum of the foot was abolished at the first was abolished at the first test after the surgery and recovered slowly during the follow- test after the surgery and recovered slowly during the follow-up to close to normal levels, up to close to normal levels, early in proximal (p < 0.0001) (Figure 1D) and middle (p < early in proximal (p < 0.0001) (Figure 1D) and middle (p < 0.05) (Figure 1E) sites, likely 0.05) (Figure 1E) sites, likely due to collateral reinnervation. In contrast, the reflex in the due to collateral reinnervation. distal site showed a returnIn contrast, the reflex of response in thewith compatible distal site showed peroneal a return reinnervation, of reaching response compatible withinperoneal full recovery reinnervation, all the sheep of AG5 group reaching at the endfullofrecovery the studyin(pall the sheep < 0.001 of at vs. values AG5 group at30the end and days), of the in study (p