Reflex-based grasping, skilled forelimb reaching, and electrodiagnostic evaluation for comprehensive analysis of functional recovery-The 7-mm rat median nerve gap repair model revisited

Abstract

Introduction: The rat median nerve injury and repair model gets increasingly important for research on novel bioartificial nerve grafts. It allows follow-up evaluation of the recovery of the forepaw functional ability with several sensitive techniques. The reflex-based grasping test, the skilled forelimb reaching staircase test, as well as electrodiagnostic recordings have been described useful in this context. Currently, no standard values exist, however, for comparison or comprehensive correlation of results obtained in each of the three methods after nerve gap repair in adult rats.

Methods: Here, we bilaterally reconstructed 7-mm median nerve gaps with autologous nerve grafts (ANG) or autologous muscle-in-vein grafts (MVG), respectively. During 8 and 12 weeks of observation, functional recovery of each paw was separately monitored using the grasping test (weekly), the staircase test, and noninvasive electrophysiological recordings from the thenar muscles (both every 4 weeks). Evaluation was completed by histomorphometrical analyses at 8 and 12 weeks postsurgery.

Results: The comprehensive evaluation detected a significant difference in the recovery of forepaw functional motor ability between the ANG and MVG groups. The correlation between the different functional tests evaluated precisely displayed the recovery of distinct levels of forepaw functional ability over time.

Conclusion: Thus, this multimodal evaluation model represents a valuable preclinical model for peripheral nerve reconstruction approaches.

Keywords: electrophysiology; grasping test; rat median nerve; staircase test.

Figure 1

Photograph of the grasping test device and picture details taken from video sequences as an example of the grasping ability categorization. (a) The grasping device was mainly composed of the digital force transducer (indicated by #) that was attached on a heavy base plate and cable connected to a Grip Strength Test Meter (indicated by *) for measurement of the forelimb grip force in [g] on the one side while on the other side the enhanced grasping frame was screwed. (b–d) The animal was held by its tail and was gently passed over the enhanced grasping frame until it touched the upper metal bar (b). Due to the grasping reflex, the animal immediately grasps the bar in case of proper innervation of the finger flexors (d). Then, the animal was slowly lifted while it was pulling the grasping frame in the indicated direction (arrow in a). In the first weeks following surgery, animals were not able to grasp the metal bar after touching it (b). After a while, reinnervation led to regained finger flexion even though in the beginning, this usually did not occur at the same time in both paws (c). That is why video analysis is an essential tool to evaluate bilaterally treated animals. The white tape seen in all pictures was utilized to avoid holding the bar via wrist flexion

Figure 2

Photographs demonstrating the ventral view of electrode positioning during noninvasive electrophysiological recordings and of the magnified palmar view of the right paw to clarify exact positioning of the recording electrode. (a) Proximal stimulation was achieved by introducing active (4a) and passive (4p) stimulation electrodes close together in the axillary region while distal stimulation was performed by placing active (3a) and passive (3p) stimulation electrodes close together at the elbow. The ground electrode (5) was subcutaneously introduced above the sternum. (b) As indicated by the black arrows, the recording electrode (2) was positioned at the thenar muscle between the rudimentary thumb (I) and the medial walking pad (a), while the reference electrode (1) was placed in the finger tip of the second digit (II)

Figure 3

Classification of the individual paw usage abilities based on the weekly performed reflex‐based grasping test revealed the onset and progression of functional motor recovery over 12 weeks postsurgery. With the help of video analysis, an earlier onset of functional recovery was found in autologous nerve graft–reconstructed animals leading to complete functional recovery by the end of observation. Functional recovery of muscle‐in‐vein graft repaired animals was delayed and incomplete at 12 weeks postsurgery (n = 16 at 4 and 8 weeks postsurgery; n = 8 at 12 weeks postsurgery). No statistical evaluation was applied. Values are given as percentages related to all evaluated paws in the particular group

Figure 4

Quantitative results of the staircase test training phase (a) and its following application to evaluate functional motor recovery by means of fine motor skills over 12 weeks postsurgery (b). (a) During 10 consecutive days of training, paw usage abilities constantly improved leading to a plateau phase (from day 7 onward) in each paw by the end of training (n = 16 paws per group). The last three achievements (indicated by filled symbols) were taken into account to calculate the healthy state mean reference value for each individual paw. Significant paw preference was found in most animals leading to a better performance with the right paw. Two‐way ANOVA followed by Sidak's multiple comparison was applied to examine significant differences (*p < .05, **p < .01, ***p < .001 vs. contralateral paw). Values are given as mean ± SEM. (b) The staircase test was applied to monitor recovery of motivation‐induced fine motor skills and demonstrated a significant increase in both groups over time without significant differences between the groups (autologous nerve graft [ANG]: n = 16 at 4 and 8 weeks postsurgery; MVGs (muscle‐in‐vein grafts): n = 14 paws at 4 and 8 weeks postsurgery, one animal (two paws) had to be excluded due to missing participation during the training phase; ANG and MVG: n = 8 paws at 12 weeks postsurgery). Two‐way ANOVA followed by Tukey's multiple comparison was applied to examine significant differences (***p < .001 vs. 4 weeks postsurgery). Values are displayed as median ± range and given as percentages related to the previously set individual healthy state reference mean values resulting in a healthy state baseline at 100%

Figure 5

Quantitative results of the electrodiagnostic recordings from the thenar muscle depicting motor recovery over 12 weeks postsurgery. Evoked compound muscle action potentials (CMAPs) were recorded to evaluate an amplitude area over time as common indicator related to motor recovery. While nerve reconstruction with autologous nerve grafts resulted in a significant increase, reconstruction with muscle‐in‐vein grafts led to no significant improvement (n = 16 paws evaluated per group at 4 and 8 weeks postsurgery; n = 8 paws evaluated at 12 weeks postsurgery). The horizontal continuous line indicates the healthy nerve reference mean value recorded presurgically from n = 16 animals. Two‐way ANOVA followed by Tukey's multiple comparison was applied to examine significant differences (*p < .05, ***p < .001 vs. 4 weeks postsurgery; ^$$$ p < .001 vs. 8 weeks postsurgery; ^### p < .001 as linked). Values are given as median ± range

Figure 6

Macroscopic appearance of the sutured grafts during reconstruction surgery (a, b) and of the regenerated tissue upon tissue harvest at 12 weeks postsurgery (c–e). To bridge the previously transected median nerves, either a autologous nerve graft (ANG) (a) or a muscle‐in‐vein graft (MVG) (b) was inserted in a length of 7 mm and subsequently sutured with three stitches at each nerve end (ds = distal suture, ps = proximal suture). Scale paper (millimeter scale) at the bottom of the photomicrographs indicates the distance between the sutures. (c) ANGs showed an unaltered macroscopical appearance upon tissue harvest at 12 weeks postsurgery while (d, e) obvious neuroma formation was found at the proximal suture sites of all MVGs 12 weeks postsurgery (indicated by *; () encircle sutures that were added posttissue harvest to serve as identification marks for immunohistology). While ANGs and MVGs with a good regenerative outcome kept their original length of 7 mm (c, d), MVGs that were stretched up to 10 mm in length (E) led to incomplete functional recovery, although upon following nerve morphometrical evaluation, single axons were found in the distal nerve segments which qualified to induce evocable compound muscle action potentials in electrodiagnostic measurements. Explanted specimens have been placed on scale paper (millimeter scale) in order to indicate the dimensions

Figure 7

Representative photomicrographs of consecutive cross‐sections through a healthy median nerve (a, b) compared to the regenerated tissue in the middle of either autologous nerve grafts (ANGs) (c, d) or muscle‐in‐vein grafts (MVGs) (e, f) 8 weeks postsurgery. (a, c, e) Immunohistological evaluation displays the immunodetection of healthy and regenerated axons (NF200, green), myelin profiles (MBP, red), and nuclear staining (DAPI, blue). (b, d, f) HE staining allows the discrimination of different tissue types. Healthy nerves revealed dense nerve tissue surrounded only by the epineurium (a, b). ANGs showed a less compact nerve tissue surrounded by a thicker layer of loose connective tissue that had formed around the epineurial layer (c, d). In MVGs, nerve tissue had mainly formed inside the vein walls (indicated by the white arrow) while the regrown nerve tissue has not completely replaced the muscle tissue (indicated by the *) until 8 weeks postsurgery (e, f). Eventually huge cell accumulations were found on the outsides of the vein walls (see box for magnification). (e) In the outer area of the section immunostaining gave some unspecific signals. White scale bars display 150 μm

Figure 8

Representative high‐resolution pictures of toluidine blue‐stained semithin cross‐sections showing a healthy distal nerve segment (a) compared to distal nerve segments of reconstructed median nerves (b–d) 12 weeks postsurgery. Images indicate different nerve fiber densities following the application of either autologous nerve grafts (b) or muscle‐in‐vein grafts (MVGs) (c, d) compared to healthy nerve samples (a). Whereas all shown samples led to an evocable compound muscle action potential recorded from the thenar muscle, MVGs with a low nerve fiber density (c; yellow arrows highlight single myelinated axons) did not result in successful participation of the affected forelimbs in the behavioral tests until 12 weeks postsurgery. White scale bars display 15 μm

Figure 9

Quantitative results of the nerve morphometrical analysis of healthy nerve segments compared to distal nerve segments of reconstructed median nerves at 8 and 12 weeks postsurgery. Horizontal continuous lines indicate corresponding healthy nerve mean values (n = 3). (a–c) Histograms representing the cross‐sectional area of the whole distal nerve segment (a), the total number of myelinated axons (b), and the resulting nerve fiber density (c) (n = 8). (d–g) Histograms depicting nerve regeneration‐related size parameters: axon diameter (d), fiber diameter (e), g‐ratio (f), and myelin thickness (g) (n = 4). Two‐way ANOVA followed by Tukey's multiple comparison was applied to examine significant differences (*p < .05, ***p < .001 as linked). Values are given as mean ± SEM. (h–j) Histograms illustrating diameter frequency distributions of myelinated nerve fibers 8 weeks (H) and 12 weeks (j) postsurgery obtained by pooling all measured values (n = 320 analyzed axons). The fiber diameter distribution of healthy nerve samples is given as a curve in the background (n = 240 analyzed axons). Values are expressed as mean percentages

Figure 10

Schematic drawing of the progress in motor recovery over time as detectable with the three different periodically applied noninvasive functional evaluation methods