Figure 1.
Representative pictures of injury zones (arrows) are shown for each of the femoral and sciatic nerve experimental groups, proximal ends to the left. SHAM – nerve exposure and suture marking only; CRUSH – simple 30-second jeweler’s forceps crush; MN – single 3-second compression using sub-transection force; MN+50g – single MN compression combined with 50g traction force; (MN+50g)x2 – two MN+50g injuries made in tandem; TRANSECTION – sharp transection without repair; TRANSECTION+REPAIR – intra-tubular repair of the transected femoral nerves; NEGATIVE CONTROL – transection, capping and back-reflection of sciatic nerve ends as negative control.
Figure 2.
Selective sciatic motor neuron labeling for assessment of relative axonal misdirection.
For demonstration purposes, representative coronal cut hemi-cord examples (stacks of all spinal cord sections of a single animal from each group) show the disorganization of labeled neurons after more severe nerve injuries, compared to Shams (stack of all 6 sham cords used to define the reference boundaries). FB (blue) or Di-I (yellow) labeled cells outside the reference bounds have axons misdirected respectively to the MG or sural nerves. Caudal sciatic pool boundaries were aligned to overlay the Shams group pool reference grid for the determination of motor neurons with misdirected axons (500µm scale bar).
Figure 3.
Neuroma formation in femoral nerve NIC groups.
Representative longitudinal femoral nerve sections of the experimental NIC groups, with magnified areas demonstrating extrafascicular regeneration on the right. MNf injury zones showed the least, and (MN+50g)x2f (proximal injury) the most prominent NIC features. Proximal ends to the left, NF 200 in green, Rhodamine Phalloidin (f-actin) in red (250µm scale bar).
Figure 4.
Femoral nerve motor division histomorphometry.
Representative semi-thin transverse sections of the motor division of each femoral nerve group stained with toluidine blue. A) Fiber diameters in two NIC groups were not significantly different from Crushf (n=6), unlike the (MN+50g)x2f (n=5), Transectionf (n=5) and Transection+Repairf (n=6) groups (*), although all were different from Shamf (**). B) Percentage neural tissue in the (MN+50g)x2f group showed statistically significant differences compared to the Shamf (n=6) and Crushf groups, similar to the Transectionf and Transection+Repairf groups. MNf (n=3); MN+50gf (n=4); 20µm scale bar (* and ** p<0.05).
Figure 5.
Femoral nerve motor neuron labeling results.
Results of motor neuron labeling by FB (A) and Di-I (B) application to femoral nerve motor and cutaneous divisions, respectively. Double-labeled motor neurons (C) are illustrated by the arrows (merge A+B). The total MN counts (FB + Di-I minus double label) represent the overall degree of attrition of motor neurons that regenerated axons beyond the injury zone. The total Crushf (n=6) group counts dominated over (MN+50g)x2f (n=4) and Transection+Repairf (n=5) groups with statistical significance (D). FB cell counts of the Shamf (n=5) and Crushf groups showed statistically significant differences compared to the (MN+50g)x2f, Transectionf (n=5) and Transection+Repairf groups, indicating significant motor pathway attrition in the latter groups (E). Percentage motor axons misdirected to the cutaneous division (= Di-I labeled/ Total count x100) was the highest in the Transectionf group and together with the (MN+50g)x2f group, had statistically significant differences from the Shamf, Crushf, MNf (n=6) and MN+50gf (n=6) groups. The Transectionf group had also significantly more misdirection compared to the Transection+Repairf group (F); 100µm scale bar (*p<0.05;**p<0.01).
Figure 6.
Sciatic experiment muscle weight and motor neuron labeling results.
Tibialis anterior muscle weights demonstrated no significant differences between the NIC groups or from Crushs and Shams groups. Transections and Negative Controls groups showed statistically significant differences from other groups (A) (n=6 per group). Statistically significant attrition of motor neurons with axons that regenerated into the MG nerve was demonstrated in the Transections group, compared to Shams and Crushs groups (B) (n=6 per group). Relative percentage of axonal misdirection to the MG and sural nerves were calculated by dividing the counted cells “out”-side the reference boundaries by the “total” counts (e.g. %misdirection to sural = Di-I-out/Di-I-total x100). Transections (n=5) injuries demonstrated the most misdirection and the same trend was found among the NIC injury groups with MG and sural nerve assessments. Average of MG and sural results shown (C) (n=6 per other groups). (*p<0.05,**p<0.001).
Figure 7.
Behavioral results and correlations with axonal misdirection and attrition.
In sciatic nerve groups, skilled locomotion was assessed with the ladder-rung task by determining injured limb slip ratios up to 12 weeks. The Crushs group recovered to baseline, but the Transections and Negative Controls groups showed no significant recovery. Despite variable deficits ranging from minimal to extreme in the NIC injury groups, the MNs and (MN+50g)x2s groups still demonstrated statistically significant differences from Crushs at 12 weeks (A) (n=6 per group). More significant than the group results, were the good correlation between individual animal final functional outcomes and the average percentage of misdirection (r2= 0.67) and FB labeled motor neuron count (attrition of motor neurons with axons to MG nerve) (r2= 0.69) respectively (B). A better correlation was found between the final functional deficit and the misdirection/regeneration quotient (MRQ = Average misdirection (%)/ MG motor neurons (n); r2=.76), which combines the relative contribution of both these factors (C).
Figure 8.
The applied force to severity of nerve injury relationship demonstrating the NIC spectrum and NIC window.
Based on the data presented, we propose that the degree of functional deficit that follows traumatic peripheral nerve injuries is dependent on the disruption of the internal nerve architecture of a particular nerve. Sunderland grade 3 and 4 injuries are replaced by the NIC spectrum. Minor variations of force on the hypothetical steep slope within the NIC window may have a dramatic effect on the injury severity and resultant functional recovery.