Artur S.P. Varejão

Functional evaluation of peripheral nerve regeneration in the rat : walking track analysis

The experimental model of choice for many peripheral nerve investigators is the rat. Walking track analysis is a useful tool in the evaluation of functional peripheral nerve recovery in the rat. This quantitative method of analyzing hind limbs performance by examining footprints, known as the sciatic function index (SFI), has been widely used to quantify functional recovery from sciatic nerve injury in a number of different injury models, although some limitations of the SFI has been questioned by several authors. This article is designed to offer the peripheral nerve investigator a noninvasive method to evaluate quantitatively the integrated motor recovery in experimental studies.

Methods for the experimental functional assessment of rat sciatic nerve regeneration

Abstract In experimental peripheral nerve studies, the rat sciatic nerve model is widely used to examine functional changes after different surgical repairs or pharmacological treatments, following nerve injury. 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, therefore, this article describes in more detail the method to obtain and measure the walking tracks in order to calculate the sciatic functional index (SFI). However, it is important to note that the validity of the SFI has been questioned by several researchers. In addition, the present review includes other traditional tests described in the experimental peripheral nerve literature regarding the rate of return of motor function and sensation, such as the extensor postural thrust (EPT), nociceptive function, and the gastrocnemius-soleus weight parameters. In the last decade, several authors have designed a series of sensitive quantitative methods to assess the recovery of hind limb locomotor function using computerized rat gait analysis. This study aims to review kinematic measures that can be gathered with this technology, including calculation of sciatic functional index, gait-stance duration, ankle kinematics and toe out angle (TOA). A combination of tests, each examining particular components of recovered sensorimotor function is recommended for an overall assessment of rat sciatic nerve regeneration.

Use of hybrid chitosan membranes and N1E-115 cells for promoting nerve regeneration in an axonotmesis rat model

Many studies have been dedicated to the development of scaffolds for improving post-traumatic nerve regeneration. The goal of this study was to develop and test hybrid chitosan membranes to use in peripheral nerve reconstruction, either alone or enriched with N1E-115 neural cells. Hybrid chitosan membranes were tested in vitro, to assess their ability in supporting N1E-115 cell survival and differentiation, and in vivo to assess biocompatibility as well as to evaluate their effects on nerve fiber regeneration and functional recovery after a standardized rat sciatic nerve crush injury. Functional recovery was evaluated using the sciatic functional index (SFI), the static sciatic index (SSI), the extensor postural thrust (EPT), the withdrawal reflex latency (WRL) and ankle kinematics. Nerve fiber regeneration was assessed by quantitative stereological analysis and electron microscopy. All chitosan membranes showed good biocompatibility and proved to be a suitable substrate for plating the N1E-115 cellular system. By contrast, in vivo nerve regeneration assessment after crush injury showed that the freeze-dried chitosan type III, without N1E-115 cell addition, was the only type of membrane that significantly improved posttraumatic axonal regrowth and functional recovery. It can be thus suggested that local enwrapping with this type of chitosan membrane may represent an effective approach for the improvement of the clinical outcome in patients receiving peripheral nerve surgery.

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

We have recently described the sequence of functional and morphologic changes occurring after a standardized sciatic nerve crush injury. An 8-week post-injury time was used because this end point is the far most used. Unexpectedly, both functional and morphological data revealed that animals had still not recovered to normal pre-injury levels. Therefore, the present study was designed in order to prolong the observation up to 12 weeks. Functional recovery was evaluated using sciatic functional index (SFI), static sciatic index (SSI), extensor postural thrust (EPT), withdrawal reflex latency (WRL) and ankle kinematics. In addition, quantitative morphology was carried out on regenerated nerve fibers. A full functional recovery was predicted by SFI/SSI, EPT and WRL but not all ankle kinematics parameters. Moreover, only two morphological parameters (myelin thickness/axon diameter ratio and fiber/axon diameter ratio) returned to normal values. Data presented in this paper provide a baseline for selecting the adequate end-point and methods of recovery assessment for a rat sciatic nerve crush study and suggest that the combined use of functional and morphological analysis should be recommended in this experimental model.

Toe out angle : a functional index for the evaluation of sciatic nerve recovery in the rat model

In experimental peripheral nerve studies, the rat sciatic nerve model is widely used to examine functional outcome following nerve injury and repair. A variety of evaluation methods exist in the literature, but an adequate selection continues to be a critical point for the researcher. Rats with sciatic nerve injury typically ambulate with an external rotation of the foot. A new functional assessment instrument, the toe out angle (TOA) is quantified using computerized gait analysis. We compared Sciatic Functional Index (SFI) with TOA parameter after peripheral nerve transection and entubulation repair. We found a good correlation between SFI and TOA measurements in terms of predicting functional recovery. Moreover, the TOA provides information on the biomechanical consequences of the external rotation of the foot in the stance phase of walking.

Motion of the foot and ankle during the stance phase in rats

Computerized analysis of rat gait is becoming an invaluable technique used by some peripheral nerve investigators for the evaluation of function. In this article we describe the use of a biomechanical model of the foot and ankle that allows a quantitative assessment and description of the ankle angle, reflecting plantarflexion and dorsiflexion during the stance phase of gait. Kinematic data of 144 trial walks from 36 normal rats were recorded with a high‐speed digital image camera at 225 images per second. The ankle angular changes associated with the specific temporal events of foot placement on the ground through the stance phase were assessed. The information obtained was used to propose a new subdivision of the stance phase in the rat into three major components. This approach will provide a helpful research tool to analyze gait data that rely on the accurate determination of spatiotemporal foot events.

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.

Ankle kinematics to evaluate functional recovery in crushed rat sciatic nerve

Peripheral nerve researchers frequently use the rat sciatic nerve crush model in order to test different therapeutic approaches. The purpose of this study was to determine the sequence of changes after an axonotmetic injury by means of a biomechanical model of the foot and ankle, and compare them with walking track analysis, over a fixed period of time. A kinematic analysis program was used to acquire ankle motion data for further analysis. Although repeated measures analysis of variance showed significant cumulative changes induced by the crush lesion for both ankle kinematic parameters and sciatic functional index, post‐hoc multiple comparisons by the Student‐Neuman‐Keuls test revealed significant differences between week 0 and week 8 only for ankle kinematics. These results are of importance in showing the superiority of ankle kinematics in detecting small biomechanical deficits related to hyperexcitability of the plantarflexor muscles, in contrast with walking track analysis, which showed full motor functional recovery 8 weeks after the crush lesion. Muscle Nerve 27: 706–714, 2003

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 crush injury of the rat median nerve

The availability of effective experimental models for investigating nerve regeneration and designing new strategies for promoting this unique repair process is important. The aim of this study was to standardize a rat median nerve crush injury model using a non-serrated clamp exerting a compression force of 17.02 MPa for a duration of 30s. Results showed that functional recovery, evaluated by grasping test, was already detectable at day-12 and progressively increased until day-28 after which animal performance plateaued until the end of testing (day-42), reaching a range of 75-80% of pre-operative values. Morphological analysis on the median nerve segments, distal to the crush lesion, which were withdrawn at the end of the experiment showed that regenerated nerve fibers are significantly more numerous and densely packed; they are also smaller and have a thinner myelin sheath compared to controls. Together, these results provide a baseline characterization of the crush median nerve injury experimental model for its employment in the investigation of nerve regeneration research, especially when a reproducible regeneration process is required, such as for the study of biological mechanisms of peripheral nerve fiber regeneration or development of new therapeutic agents for promoting posttraumatic nerve repair.