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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Contribution of cutaneous inputs from the hindpaw to the control of locomotion in rats.

The influence of reduced feedback from the cutaneous receptors in the hindpaw in rat locomotion is still unclear. To evaluate this question, we conducted a detailed hindlimb kinematic analysis in animals, which suffered complete loss of thermal sensation. Two-dimensional hindlimb kinematics, temporal and spatial measurements, and walking track analysis were performed in rats before and during hypothermic anesthesia. The walking velocity, duration of the step cycle and stance phase, and stride length between the two testing conditions were statistically indistinguishable. Swing phase duration was significantly decreased during sensory loss. Analysis of angular motion revealed an increased hip and knee extension and an ankle joint with increased flexion during the step cycle under plantar anesthesia. Also after plantar cooling, the hip and knee angular velocity was significantly affected along the step cycle. The remarkably geometric similarity of the angle-angle plots obtained in our experiments reflected an interjoint coordination; however, the interpretation of the cyclogram perimeter revealed a larger excursion by the ankle and hip in their respective joint spaces in rats deprived of sensation. Examination of the horizontal position of the ankle with respect to the hip and the extension before toe-off revealed no major changes, whereas, there was a slight decrease in distance of the hip to the ground during sensory loss. Also, the walking tracks revealed a significant functional deficit following reduced cutaneous information of the plantar aspect of the hindpaw. We therefore conclude that sensory feedback from the hindpaw is important in the maintenance of normal rat locomotion.

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.

Effect of skin movement on the analysis of hindlimb kinematics during treadmill locomotion in rats.

In rat gait kinematics, the method most frequently used for measuring hindlimb movement involves placing markers on the skin surface overlying the joints being analyzed. Soft tissue movement around the knee joint has been considered the principle source of error when estimating hindlimb joint kinematics in rodents. However, the motion of knee marker was never quantified, nor the different variations in joint angle associated with this gait analysis system. The purpose of this study was two-fold. The first purpose was to expand upon the limited pool of information describing the effect of soft tissue movement over the knee upon the angular positions of the hip, knee and ankle of rats during treadmill locomotion. Secondly, it was a goal of this study to document the magnitude of the skin displacement when using markers that were attached superficially to the knee joint. This was examined by comparing the hindlimb kinematics in sagittal plane during treadmill locomotion determined from the marker attached to the knee and when the knee position was determined indirectly by computer analysis. Results showed that there is a considerable skin movement artefact which propagates to knee joint position and hindlimb kinematics estimates. It was concluded that these large errors can decrease data reliability in the research of rat gait analysis.

A comparison analysis of hindlimb kinematics during overground and treadmill locomotion in rats.

The convenience of the motor-driven treadmill makes it an attractive instrument for investigating rat locomotion. However, no data are available to indicate whether hindlimb treadmill kinematic findings may be compared or generalized to overground locomotion. In this investigation, we compared overground and treadmill locomotion for differences in the two-dimensional angular kinematics and temporal and spatial measurements for the hindlimb. Ten female rats were evaluated at the same speed for natural overground and treadmill walking. The walking velocity, swing duration and stride length were statistically indistinguishable between the two testing conditions. Significant differences were found between overground and treadmill locomotion for step cycle duration and stance phase duration parameters. During the stance phase of walking, the angular movement of the hip, knee and ankle joints were significantly different in the two conditions, with greater flexion occurring on the overground. Despite this, the sagittal joint movements of the hindlimb were similar between the two walking conditions, with only three parameters being significantly different in the swing. Hip height and angle-angle cyclograms were also only found to display subtle differences. This study suggests that reliable kinematic measurements can be obtained from the treadmill gait analysis in rats.

Functional and morphological assessment of a standardized rat sciatic nerve crush injury with a non-serrated clamp.

Peripheral nerve researchers frequently use the rat sciatic nerve crush as a model for axonotmesis. Unfortunately, studies from various research groups report results from different crush techniques and by using a variety of evaluation tools, making comparisons between studies difficult. The purpose of this investigation was to determine the sequence of functional and morphologic changes after an acute sciatic nerve crush injury with a non-serrated clamp, giving a final standardized pressure of p = 9 MPa. Functional recovery was evaluated using the sciatic functional index (SFI), the extensor postural thrust (EPT) and the withdrawal reflex latency (WRL), before injury, and then at weekly intervals until week 8 postoperatively. The rats were also evaluated preoperatively and at weeks 2, 4, and 8 by ankle kinematics, toe out angle (TOA), and gait-stance duration. In addition, the motor nerve conduction velocity (MNCV) and the gastrocnemius-soleus weight parameters were measured just before euthanasia. Finally, structural, ultrastructural and histomorphometric analyses were carried out on regenerated nerve fibers. At 8 weeks after the crush injury, a full functional recovery was predicted by SFI, EPT, TOA, and gait-stance duration, while all the other parameters were still recovering their original values. On the other hand, only two of the histomorphometric parameters of regenerated nerve fibers, namely myelin thickness/axon diameter ratio and fiber/axon diameter ratio, returned to normal values while all other parameters were significantly different from normal values. The employment of traditional methods of functional evaluation in conjunction with the modern techniques of computerized analysis of gait and histomorphometric analysis should thus be recommended for an overall assessment of recovery in the rat sciatic nerve crush model.

Use of skeletal muscle tissue in peripheral nerve repair: review of the literature.

The management of peripheral nerve injury continues to be a major clinical challenge. The most widely used technique for bridging defects in peripheral nerves is the use of autologous nerve grafts. This technique, however, necessitates a donor nerve and corresponding deficit. Many alternative techniques have thus been developed. The use of skeletal muscle tissue as graft material for nerve repair is one example. The rationale regarding the use of the skeletal muscle tissue technique is the availability of a longitudinally oriented basal lamina and extracellular matrix components that direct and enhance regenerating nerve fibers. These factors provide superiority over other bridging methods as vein grafts or (non)degradable nerve conduits. The main disadvantages of this technique are the risk that nerve fibers can grow out of the muscle tissue during nerve regeneration, and that a donor site is necessary to harvest the muscle tissue. Despite publications on nerve conduits as an alternative for peripheral nerve repair, autologous nerve grafting is still the standard care for treatment of a nerve gap in the clinical situation; however, the use of the skeletal muscle tissue technique can be added to the surgeon's arsenal of peripheral nerve repair tools, especially for bridging short nerve defects or when traditional nerve autografts cannot be employed. This technique has been investigated both experimentally and clinically and, in this article, an overview of the literature on skeletal muscle grafts for bridging peripheral nerve defects is presented.

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.