Peripheral Nerve Injury Treatments and Advances: One Health Perspective.

Peripheral nerve injuries (PNI) can have several etiologies, such as trauma and iatrogenic interventions, that can lead to the loss of structure and/or function impairment. These changes can cause partial or complete loss of motor and sensory functions, physical disability, and neuropathic pain, which in turn can affect the quality of life. This review aims to revisit the concepts associated with the PNI and the anatomy of the peripheral nerve is detailed to explain the different types of injury. Then, some of the available therapeutic strategies are explained, including surgical methods, pharmacological therapies, and the use of cell-based therapies alone or in combination with biomaterials in the form of tube guides. Nevertheless, even with the various available treatments, it is difficult to achieve a perfect outcome with complete functional recovery. This review aims to enhance the importance of new therapies, especially in severe lesions, to overcome limitations and achieve better outcomes. The urge for new approaches and the understanding of the different methods to evaluate nerve regeneration is fundamental from a One Health perspective. In vitro models followed by in vivo models are very important to be able to translate the achievements to human medicine.

A Comparison of Two-Dimensional and Three-Dimensional Techniques for Kinematic Analysis of the Sagittal Motion of Sheep Hindlimbs During Walking on a Treadmill.

Compared to rodents, sheep offer several attractive features as an experimental model for testing different medical and surgical interventions related to pathological gait caused by neurological diseases and injuries. To use sheep for development of novel treatment strategies in the field of neuroscience, it is key to establish the relevant kinematic features of locomotion in this species. To use sheep for development of novel treatment strategies in the field of neuroscience, it is crucial to understand fundamental baseline characteristics of locomotion in this species. Despite their relevance for medical research, little is known about the locomotion in the ovine model, and next to nothing about the three-dimensional (3D) kinematics of the hindlimb. This study is the first to perform and compare two-dimensional (2D) and 3D hindlimb kinematics of the sagittal motion during treadmill walking in the ovine model. Our results show that the most significant differences took place throughout the swing phase of the gait cycle were for the distal joints, ankle and metatarsophalangeal joint, whereas the hip and knee joints were much less affected. The results provide evidence of the inadequacy of a 2D approach to the computation of joint kinematics in clinically normal sheep during treadmill walking when the interest is centered on the hoof's joints. The findings from the present investigation are likely to be useful for an accurate, quantitative and objective assessment of functionally altered gait and its underlying neuronal mechanisms and biomechanical consequences.

Establishment of a Sheep Model for Hind Limb Peripheral Nerve Injury: Common Peroneal Nerve.

Thousands of people worldwide suffer from peripheral nerve injuries and must deal daily with the resulting physiological and functional deficits. Recent advances in this field are still insufficient to guarantee adequate outcomes, and the development of new and compelling therapeutic options require the use of valid preclinical models that effectively replicate the characteristics and challenges associated with these injuries in humans. In this study, we established a sheep model for common peroneal nerve injuries that can be applied in preclinical research with the advantages associated with the use of large animal models. The anatomy of the common peroneal nerve and topographically related nerves, the functional consequences of its injury and a neurological examination directed at this nerve have been described. Furthermore, the surgical protocol for accessing the common peroneal nerve, the induction of different types of nerve damage and the application of possible therapeutic options were described. Finally, a preliminary morphological and stereological study was carried out to establish control values for the healthy common peroneal nerves regarding this animal model and to identify preliminary differences between therapeutic methods. This study allowed to define the described lateral incision as the best to access the common peroneal nerve, besides establishing 12 and 24 weeks as the minimum periods to study lesions of axonotmesis and neurotmesis, respectively, in this specie. The post-mortem evaluation of the harvested nerves allowed to register stereological values for healthy common peroneal nerves to be used as controls in future studies, and to establish preliminary values associated with the therapeutic performance of the different applied options, although limited by a small sample size, thus requiring further validation studies. Finally, this study demonstrated that the sheep is a valid model of peripheral nerve injury to be used in pre-clinical and translational works and to evaluate the efficacy and safety of nerve injury therapeutic options before its clinical application in humans and veterinary patients.

Combined Use of Chitosan and Olfactory Mucosa Mesenchymal Stem/Stromal Cells to Promote Peripheral Nerve Regeneration In Vivo.

Peripheral nerve injury remains a clinical challenge with severe physiological and functional consequences. Despite the existence of multiple possible therapeutic approaches, until now, there is no consensus regarding the advantages of each option or the best methodology in promoting nerve regeneration. Regenerative medicine is a promise to overcome this medical limitation, and in this work, chitosan nerve guide conduits and olfactory mucosa mesenchymal stem/stromal cells were applied in different therapeutic combinations to promote regeneration in sciatic nerves after neurotmesis injury. Over 20 weeks, the intervened animals were subjected to a regular functional assessment (determination of motor performance, nociception, and sciatic indexes), and after this period, they were evaluated kinematically and the sciatic nerves and cranial tibial muscles were evaluated stereologically and histomorphometrically, respectively. The results obtained allowed confirming the beneficial effects of using these therapeutic approaches. The use of chitosan NGCs and cells resulted in better motor performance, better sciatic indexes, and lower gait dysfunction after 20 weeks. The use of only NGGs demonstrated better nociceptive recoveries. The stereological evaluation of the sciatic nerve revealed identical values in the different parameters for all therapeutic groups. In the muscle histomorphometric evaluation, the groups treated with NGCs and cells showed results close to those of the group that received traditional sutures, the one with the best final values. The therapeutic combinations studied show promising outcomes and should be the target of new future works to overcome some irregularities found in the results and establish the combination of nerve guidance conduits and olfactory mucosa mesenchymal stem/stromal cells as viable options in the treatment of peripheral nerves after injury.

Rat Olfactory Mucosa Mesenchymal Stem/Stromal Cells (OM-MSCs): A Characterization Study.

Stem/stromal cell-based therapies are a branch of regenerative medicine and stand as an attractive option to promote the repair of damaged or dysfunctional tissues and organs. Olfactory mucosa mesenchymal stem/stromal cells have been regarded as a promising tool in regenerative therapies because of their several favorable properties such as multipotency, high proliferation rate, helpful location, and few associated ethical issues. These cells are easily accessible in the nasal cavity of most mammals, including the rat, can be easily applied in autologous treatments, and do not cope with most of the obstacles associated with the use of other stem cells. Despite this, its application in preclinical trials and in both human and animal patients is still limited because of the small number of studies performed so far and to the nonexistence of a standard and unambiguous protocol for collection, isolation, and therapeutic application. In the present work a validation of a protocol for isolation, culture, expansion, freezing, and thawing of olfactory mucosa mesenchymal stem/stromal cells was performed, applied to the rat model, as well as a biological characterization of these cells. To investigate the therapeutic potential of OM-MSCs and their eventual safe application in preclinical trials, the main characteristics of OMSC stemness were addressed.

Kinematic and kinetic gait analysis to evaluate functional recovery in thoracic spinal cord injured rats.

The recovery of walking function following spinal cord injury (SCI) is of major importance to patients and clinicians. In experimental SCI studies, a rat model is widely used to assess walking function, following thoracic spinal cord lesion. In an effort to provide a resource which investigators can refer to when seeking the most appropriate functional assay, the authors have compiled and categorized the behavioral assessments used to measure the deficits and recovery of the gait in thoracic SCI rats. These categories include kinematic and kinetic measurements. Within this categorization, we discuss the advantages and disadvantages of each type of measurement. The present review includes the type of outcome data that they produce, the technical difficulty and the time required to potentially train the animals to perform them, and the need for expensive or highly specialized equipment. The use of multiple kinematic and kinetic parameters is recommended to identify subtle deficits and processes involved in the compensatory mechanisms of walking function after experimental thoracic SCI in rats.

Kinematic patterns for hindlimb obstacle avoidance during sheep locomotion.

Functional recovery following general nerve reconstruction is often associated with poor results. Comparing to rat and mice experimental studies, there are much fewer investigations on nerve regeneration and repair in the sheep, and there are no studies on this subject using gait analysis in the sheep model as an assessment tool. Additionally, this is the first study evaluating obstacle negotiation and the compensatory strategies that take place at each joint in response to the obstacle during locomotion in the sheep model. This study aims to get kinematic data to serve as a template for an objective assessment of the ankle joint motion in future studies of common peroneal nerve (CP) injury and repair in the ovine model. Our results show that a moderately high obstacle set to 10% of the sheep's hindlimb length was associated to several spatial and temporal strategies in order to increase hoof height during obstacle negotiating. Sheep efficiently cleared an obstacle by increasing knee, ankle and metatarsophalangeal flexion during swing, whereas the hip joint is not affected. This study establishes the bounds of normal motion in the neurologically intact hindlimb when approached and cleared an obstacle and provides baseline data for further studies of peripheral nerve research in the ovine model.

The Nasal Cavity of the Rat and Mouse-Source of Mesenchymal Stem Cells for Treatment of Peripheral Nerve Injury.

The nasal cavity performs several crucial functions in mammals, including rodents, being involved in respiration, behavior, reproduction, and olfaction. Its anatomical structure is complex and divided into several regions, including the olfactory recess where the olfactory mucosa (OM) is located and where the capture and interaction with the environmental odorants occurs. Among the cells of this region are the OM mesenchymal stem cells (MSCs), whose location raises the possibility that these cells could be involved in the peculiar ability of the olfactory nerve to regenerate continuously throughout life, although this relationship has not yet been confirmed. These cells, like all MSCs, present functional characteristics that make them candidates in new therapies associated with regenerative medicine, namely to promote the regeneration of the peripheral nerve after injury. The availability of stem cells to be therapeutically applied essentially depends on their collection in the tissue of origin. In the case of mice and rat's OM-MSCs, knowledge about the anatomy and histology of their nasal cavity is essential in establishing effective collection protocols. The present article describes the morphological characteristics of rodent's OM and establishes an alternative protocol for access to the olfactory recess and collection of the OM. Anat Rec, 301:1678-1689, 2018. © 2018 Wiley Periodicals, Inc.

Rolipram promotes functional recovery after contusive thoracic spinal cord injury in rats.

Numerous animal model studies in the past decade have demonstrated that pharmacological elevation of cyclic AMP (cAMP) alone, or in combination with other treatments, can promote axonal regeneration after spinal cord injury. Elevation of cAMP via the phosphodiesterase 4 (PDE4) inhibitor, rolipram, decreases neuronal sensitivity to myelin inhibitors, increases growth potential and is neuroprotective. Rolipram's ability to cross the blood-brain barrier makes it a practical and promising treatment for CNS regeneration. However, several studies have questioned the efficacy of rolipram when given alone. The purpose of this investigation was to determine the effects of continuous administration of rolipram, given alone for 2 weeks, following a moderate T10 contusion injury in rat. Functional recovery was evaluated using the 21-point Basso, Beattie and Bresnahan (BBB) locomotor recovery scale and the beam walk. We used three-dimensional (3D) instrumented gait analysis to allow detailed assessment and quantification of hindlimb motion. The amount of the damaged tissue and spared white matter was estimated stereologically. Our results show that administration of rolipram following acute spinal cord contusion results in improved motor performance at each time-point. Dynamic assessment of foot motion during treadmill walking revealed a significantly decreased external rotation during the entire step cycle after 8 weeks in rolipram-treated animals. Stereological analysis revealed no significant differences in lesion volume and length. By contrast, spared white matter was significantly higher in the group treated with rolipram. Our results suggest a therapeutic role for rolipram delivered alone following acute SCI.

Effects of collagen membranes enriched with in vitro-differentiated N1E-115 cells on rat sciatic nerve regeneration after end-to-end repair.

Peripheral nerves possess the capacity of self-regeneration after traumatic injury but the extent of regeneration is often poor and may benefit from exogenous factors that enhance growth. The use of cellular systems is a rational approach for delivering neurotrophic factors at the nerve lesion site, and in the present study we investigated the effects of enwrapping the site of end-to-end rat sciatic nerve repair with an equine type III collagen membrane enriched or not with N1E-115 pre-differentiated neural cells. After neurotmesis, the sciatic nerve was repaired by end-to-end suture (End-to-End group), end-to-end suture enwrapped with an equine collagen type III membrane (End-to-EndMemb group); and end-to-end suture enwrapped with an equine collagen type III membrane previously covered with neural cells pre-differentiated in vitro from N1E-115 cells (End-to-EndMembCell group). Along the postoperative, motor and sensory functional recovery was evaluated using extensor postural thrust (EPT), withdrawal reflex latency (WRL) and ankle kinematics. After 20 weeks animals were sacrificed and the repaired sciatic nerves were processed for histological and stereological analysis. Results showed that enwrapment of the rapair site with a collagen membrane, with or without neural cell enrichment, did not lead to any significant improvement in most of functional and stereological predictors of nerve regeneration that we have assessed, with the exception of EPT which recovered significantly better after neural cell enriched membrane employment. It can thus be concluded that this particular type of nerve tissue engineering approach has very limited effects on nerve regeneration after sciatic end-to-end nerve reconstruction in the rat.

Use of chitosan scaffolds for repairing rat sciatic nerve defects.

Neurotmesis must be surgically treated by direct end-to-end suture of the two nerve stumps or by a nerve graft harvested from elsewhere in the body in case of tissue loss. To avoid secondary damage due to harvesting of the nerve graft, a tube-guide can be used to bridge the nerve gap. Previously, our group developed and tested hybrid chitosan membranes for peripheral nerve tubulization and showed that freeze-dried chitosan type III membranes were particularly effective for improving peripheral nerve functional recovery after axonotmesis. Chitosan type III membranes have about 110 microm pores and about 90% of porosity, due to the employment of freeze-drying technique. The present study aimed to verify if chitosan type III membranes can be successfully used also for improving peripheral nerve functional recovery after neurotmesis of the rat sciatic nerve. Sasco Sprague-Dawley adult rats were divided into 6 groups: Group 1: end-to-end neurorrhaphy enwrapped by chitosan membrane type III (End-to-EndChitll); Group 2: 10mm-nerve gap bridged by an autologous nerve graft enwrapped by chitosan membrane type III (Graf180degreeChitIII); Group 3: 10 mm-nerve gap bridged by chitosan type III tube-guides (GapChitIII); These 3 experimental groups were compared with 3 control groups, respectively: Group 4: 10 mm-nerve gap bridged by an autologous nerve graft (Graft180degree); Group 5: 10 mm-nerve gap bridged by PLGA 90:10 tube-guides (PLGA); Group 6: end-to-end neurorrhaphy alone (End-to-End). Motor and sensory functional recovery were evaluated throughout a healing period of 20 weeks using extensor postural thrust (EPT), withdrawal reflex latency (WRL) and ankle kinematics. Regenerated nerves withdrawn at the end of the experiment were analysed histologically. Results showed that nerve regeneration was successful in all experimental and control groups and that chitosan type III tubulization induced a significantly better nerve regeneration and functional recovery in comparison to PLGA tubulization control. Further investigation is needed to explore the mechanisms at the basis of the positive effects of chitosan type III on axonal regeneration.

The effect of gait speed on three-dimensional analysis of hindlimb kinematics during treadmill locomotion in rats.

The two-dimensional (2D) kinematic approach is by far the most popular technique in rat gait analysis. This is a simple inexpensive procedure, which requires only one camera to record the movement. However, maximal precision and accuracy of the kinematic values are expected when the experimental protocol includes a three-dimensional (3D) motion analysis methodology. Locomotor speed is a basic kinematic parameter that is often neglected in most studies of movement disorders and neurological diseases. Because locomotor speed can act as confounder for the interpretation of the obtained results we also focused our attention on the relation between speed and 3D hindlimb kinematics. Our experimental set-up consisted of a motion capture system with four CMOS cameras which allowed a non-invasive estimation of the instantaneous position of color markers in a 3D measurement volume. Data were recorded while rats walked at different treadmill speeds (30 vs 60 cm/s). For the first time we reported detailed kinematic data for the sagittal, coronal and transverse plane during treadmill locomotion in rats. Despite the overall time course patterns of the curves were identical, we found significant differences between values of joint angular motion at 30 and 60 cm/s at selected points of the step cycle. The adaptation to higher treadmill walking included greater joint angular excursions. The present report highlights the importance of walking speed when evaluating rat hindlimb kinematics during gait. Hopefully, this study will be useful in experimental data assessment when multiple gait abnormalities are expected to occur in all planes of motion.

Methylprednisolone fails to improve functional and histological outcome following spinal cord injury in rats.

Currently, methylprednisolone sodium succinate (MPSS) is the standard treatment following acute spinal cord injury (SCI) as a consequence of the results obtained from the National Acute Spinal Cord Injury Studies. However, many have questioned the efficacy of MPSS because of its marginal effects. Additionally there has been criticism of both study design and statistical interpretation. The functional consequences of experimental SCI have been assessed in many ways. The purpose of this investigation was to determine the effects of MPSS vs. saline solution (SS) following moderate T10 contusion injury in rat. Functional recovery was evaluated using the 21-point Basso, Beattie and Bresnahan (BBB) locomotor recovery scale, the inclined plane, the beam walk, footprint analysis and the horizontal ladder. To optimize the precision and accuracy of functional results we examined the locomotion on a treadmill using three-dimensional (3D) analysis. Stereology was used to estimate the amount of damaged tissue. The results of the traditional functional methods showed that administration of the NASCIS dosage of MPSS following acute spinal cord contusion did not lead to any significant differences in the functional recovery of MPSS- vs. SS-treated animals. More importantly, the results of the 3D kinematic showed that the MPSS administration did not affect the flexion/extension of the hip, knee and ankle joints during the step cycle. Finally, stereological results revealed no statistically significant differences between the two experimental groups. Altogether, our results support data previously reported by several authors, suggesting that MPSS does not lead to improved functional outcome following experimental acute SCI.

Chapter 7: Methods and protocols in peripheral nerve regeneration experimental research: part IV-kinematic gait analysis to quantify peripheral nerve regeneration in the rat.

Functional recovery is one of the primary goals of therapeutic intervention in peripheral nerve research. The number and diversity of tests which have been used to assess functional recovery after experimental interventions often makes it difficult to recommend any particular indicator of nerve regeneration. Functional assessment after sciatic nerve lesion has long been focused on walking track analysis; however, it is important to note that the validity of the sciatic functional index has been questioned by several researchers. In the last decade, several authors have designed a series of sensitive quantitative methods to assess the recovery of locomotor function using computerized rat gait analysis. The objective of the present review is to provide a helpful tool for the peripheral nerve investigator, by integrating the most important gait kinematic measures described in the literature that can be gathered with this technology.

A comparison of two-dimensional and three-dimensional techniques for the determination of hindlimb kinematics during treadmill locomotion in rats following spinal cord injury.

Of all the detrimental effects of spinal cord injury (SCI), one of the most devastating effects is the disruption of the ability to walk. Therefore, much effort has been focused on developing several methods to document the recovery of locomotor function after experimental SCI. Computerized rat gait analysis is becoming increasingly popular in the SCI research community. The two-dimensional (2D) kinematic approach is by far the most popular technique in rat gait analysis. This is a simple inexpensive procedure, which requires only one camera to record the movement. Our study included an examination of locomotion on a treadmill using 2D and three-dimensional (3D) analysis, in neurologically intact animals and following moderate T9 contusion injury. Despite the overall time course patterns of the curves were identical, we found significant differences between values of the 2D and 3D joint angular motion. In conclusion, maximal precision and accuracy of the kinematic values are expected when the experimental protocol includes a 3D motion analysis methodology. Moreover, a 2D method cannot be used to determine the external or internal rotations of the foot because this movement occurs in the transverse plane.

Use of PLGA 90:10 scaffolds enriched with in vitro-differentiated neural cells for repairing rat sciatic nerve defects.

Poly(lactic-co-glycolic acid) (PLGA) nerve tube guides, made of a novel proportion (90:10) of the two polymers, poly(L-lactide): poly(glycolide) and covered with a neural cell line differentiated in vitro, were tested in vivo for promoting nerve regeneration across a 10-mm gap of the rat sciatic nerve. Before in vivo testing, the PLGA 90:10 tubes were tested in vitro for water uptake and mass loss and compared with collagen sheets. The water uptake of the PLGA tubes was lower, and the mass loss was more rapid and higher than those of the collagen sheets when immersed in phosphate-buffered saline (PBS) solution. The pH values of immersing PBS did not change after soaking the collagen sheets and showed to be around 7.4. On the other hand, the pH values of PBS after soaking PLGA tubes decreased gradually during 10 days reaching values around 3.5. For the in vivo testing, 22 Sasco Sprague adult rats were divided into four groups--group 1: gap not reconstructed; group 2: gap reconstructed using an autologous nerve graft; group 3: gap reconstructed with PLGA 90:10 tube guides; group 4: gap reconstructed with PLGA 90:10 tube guides covered with neural cells differentiated in vitro. Motor and sensory functional recovery was evaluated throughout a healing period of 20 weeks using sciatic functional index, static sciatic index, extensor postural thrust, withdrawal reflex latency, and ankle kinematics. Stereological analysis was carried out on regenerated nerve fibers. Both motor and sensory functions improved significantly in the three experimental nerve repair groups, although the rate and extent of recovery was significantly higher in the group where the gap was reconstructed using the autologous graft. The presence of neural cells covering the inside of the PLGA tube guides did not make any difference in the functional recovery. By contrast, morphometric analysis showed that the introduction of N1E-115 cells inside PLGA 90:10 tube guides led to a significant lower number and size of regenerated nerve fibers, suggesting thus that this approach is not adequate for promoting peripheral nerve repair. Further studies are warranted to assess the role of other cellular systems as a foreseeable therapeutic strategy in peripheral nerve regeneration.

PLGA 90/10 and caprolactone biodegradable nerve guides for the reconstruction of the rat sciatic nerve.

The purpose of this study was to test in vivo two different nerve guides for promoting nerve regeneration across a 10-mm gap of the rat sciatic nerve: 1) one made of PLGA in a novel proportion (90:10) of the two polymers poly(L-lactide):poly(glycolide); 2) another made of (DL-lactide-epsilon-caprolactone) copolyester (Neurolac) tube, by comparing its healing efficacy with that of the more traditional methods of end-to-end nerve suture and autologous graft. Motor and sensory functional recovery were assessed throughout the healing period of 20 weeks, and the repaired nerves were processed for morphological and histomorphometrical analysis. Both motor and sensory functions improved significantly in all experimental nerve repaired groups. At the end of the 20-week follow-up, the end-to-end group showed better recovery of motor function when compared with the groups treated with guiding tubes. However, at this time point, the level of motor function in the Neurolac(R) and PLGA groups was similar to the one of the graft group. Nociception function also recovered faster in the end-to-end group compared with the Neurolac(R) and PLGA groups, and in this case, recovery was also delayed in the graft group. At the end of follow-up, nociception was similar in all experimental groups. Morphological and histomorphometrical analysis showed that axon regeneration occurred in both PLGA and Neurolac(R) experimental groups, with no significant differences in the total number of regenerated fibers, but disclosed a different pattern of degradation of the two types of tubes with larger biodegradation of PLGA material by the end of 20 weeks. These results suggest that both types of biomaterials are a good substrate for preparing tubular nerve guides, and their different pattern of degradation does not seem to influence the degree of nerve regeneration.

Evaluation of two biodegradable nerve guides for the reconstruction of the rat sciatic nerve.

The purpose of this study was to test in vivo two different nerve guides, one of PLGA made of a novel proportion (90:10) of the two polymers, Poly(L-lactide):Poly(glycolide), with (DL-lactide-epsilon-caprolactone) copolyester (Neurolac) tube, in promoting nerve regeneration across a 10 mm-gap of the rat sciatic nerve. Finally, end-to-end coaptation was performed. Motor and sensory functional recovery was assessed throughout the healing period of 20 weeks and the repaired nerves were processed for morphological analysis. Both motor and sensory functions improved significantly in all experimental nerve repair groups, although the rate and extent of recovery was significantly higher in the end-to-end group. No significant differences were detected in the comparison between the two types of tubes. Compatible with results of functional tests, morphological analysis showed that axon regeneration occurred in both PLGA and Neurolac experimental groups but disclosed a different pattern of degradation of the two types of tubes with larger biodegradation of PLGA material by the end of 20 weeks. These results suggest that both types of biomaterial are a good substrate for preparing tubular nerve guides and the different pattern of degradation does not seem to influence the degree of nerve regeneration.