Exacerbation of Charcot-Marie-Tooth type 2E neuropathy following traumatic nerve injury

Abstract

Charcot-Marie-Tooth disease (CMT) is the most commonly inherited peripheral neuropathy. CMT disease signs include distal limb neuropathy, abnormal gait, sensory defects, and deafness. We generated a novel line of CMT2E mice expressing hNF-L(E397K), which displayed muscle atrophy of the lower limbs without denervation, proximal reduction in large caliber axons, and decreased nerve conduction velocity. In this study, we challenged wild type, hNF-L and hNF-L(E397K) mice with crush injury to the sciatic nerve. We analyzed functional recovery by measuring toe spread and analyzed gait using the Catwalk system. hNF-L(E397K) mice demonstrated reduced recovery from nerve injury consistent with increased susceptibility to neuropathy observed in CMT patients. In addition, hNF-L(E397K) developed a permanent reduction in their ability to weight bear, increased mechanical allodynia, and premature gait shift in the injured limb, which led to increasingly disrupted interlimb coordination in hNF-L(E397K). Exacerbation of neuropathy after injury and identification of gait alterations in combination with previously described pathology suggests that hNF-L(E397K) mice recapitulate many of clinical signs associated with CMT2. Therefore, hNF-L(E397K) mice provide a model for determining the efficacy of novel therapies.

Keywords: Charcot–Marie–Tooth type 2E; Functional recovery; Gait alterations; Nerve injury; Neurofilament; Neuropathy exacerbation.

Figure 1. Functional recovery from sciatic nerve injury was reduced in hNF-L^E397K mice

Sciatic nerves were crushed at the level of the obturator tendon on four-month-old wild type, hNF-L and symptomatic hNF-L^E397K mice. Nerve injury prevented toe spread on the injured limb (A). Toe spread was quantified by measuring the distance between the first and fifth digit of the paw (B) over a time course for up to 25 days (C). Wild type and hNF-L mice fully recovered 25 days post-injury (C). Functional recovery was significantly reduced in hNF-L^E397K mice beginning at day 15(C), and hNF-L^E397K mice failed to fully recover (C). Means for each daily toe spread measurement were analyzed for overall statistical differences by two way repeated measures ANOVA with Holm-Sidak post hoc analysis., *, p< 0.05 as compared to wild type toe spread; †, p< 0.05 as compared to hNF-L toe spread.

Figure 2. Reduced Print Width after nerve crush recovery

Print Width is defined as the width of the paw from the first to the fifth digit. Print width (cm) values are reported for the left hind (A), right hind (B), left front (C), and right front (D) paws from pre-crush and post-injury analyses. Print Width failed to recover to pre-injury values for hNF-L mice only in the left hind paw (A). For hNF-L^E397K mice, Print Width was significantly decreased following recovery compared to its pre-injury values for left hind (A), left front (C), and right front (D). On the left hind (A) and right front (D) paw, hNF-L^E397K mice recovered less than hNF-L mice. The differences in means were analyzed for overall statistical differences using a two-way ANOVA with Holm-Sidak post hoc analysis. *, P < 0.05.

Figure 3. Reduced Print Length on the injured limb after recovery

Print Length is measured from the beginning of the heel to the tip of the longest digit. Print Length was significantly lower in hNF-L and hNF-L^E397K mice following recovery compared to pre-injury values (A). Additionally, hNF-L^E397K mice did not recover Print Length as well as hNF-L mice (A). Print Length values for all right hind and left front paws were unchanged after recovery (B, and C). On the right, front paw, hNF-L^E397Klengths were significantly lower compared to hNF-L lengths (D). The differences in means were analyzed for overall statistical differences using a two-way ANOVA with Holm-Sidak post hoc analysis. *, P < 0.05.

Figure 4. Max Contact Area is decreased in hind left and diagonal paw after recovery

Max Contact Area is a measure of the area contacted by a paw when the most pressure is being applied. Max Contact Area was reduced following recovery in the left hind paw of hNF-L and hNF-L^E397K mice (A). Additionally, hNF-L^E397K mice failed to recover to the same extent as hNF-L (A). Max Contact Area was unaltered in the right hind paw of hNF-L and hNF-L^E397K mice (B). Interestingly, Max Contact Area was reduced in left (C) and right (D) front paws in hNF-L^E397K mice. The differences in means were analyzed for overall statistical differences using a two-way ANOVA with Holm-Sidak post hoc analysis. *, P < 0.05.

Figure 5. Reduced Max Intensity in right front and left hind limbs post-injury

Max Intensity is a measure of the maximum light intensity of a paw print. Max Intensity correlates with the amount of pressure placed on a paw while the paw is in contact with the glass regardless of the area of contact. Max intensity failed to recover to pre-injury levels in the left hind (A) and right front (D) paws of hNF-L^E397K mice. Moreover, hNF-L^E397K animals had significantly lower Max Intensity values compared to hNF-L following recovery (A and D). Max Intensity values were unchanged in the right hind (B) and left front (C) paws. The differences in means were analyzed for overall statistical differences using a two-way ANOVA with Holm-Sidak post hoc analysis. *, P < 0.05.

Figure 6. Schematic outlining parameters measured by CatWalk

Step cycle of all paws is composed of alternating contacting (black) and non-contacting (white) phases (A). Within a step cycle, the contacting phase is referred to as the stand phase [reported as Stand (s)] and the non-contacting phase is referred to as the swing phase [reported as Swing (s)] (A). Each stand phase contains both a braking phase and propulsion phase. Maximum contact or max value of a paw roughly defines the point at which the braking phase transitions into the propulsion phase. The time it takes a paw to reach the Max Value is reported as Max Intensity At (s). Max Contact At (%) can be regarded as the point where the braking phase transitions into the propulsion phase during the Stand phase (B).

Figure 7. Reduced Max Contact At (%) in right front and left hind paws following nerve injury recovery

As Max Contact At (%) is regarded as the point where gait transitions from braking to propulsion, Max Contact At (%) is also a measure of the point within a stand phase where the maximal contact area with the walking surface is reached. hNF-L^E397K Max Contact At (%) failed to recover to pre-injury values, and was recovered less than hNF-L controls (A). Max Contact At (%) was not altered on the right hind (B) or left front (C) paws. On the right front paw, hNF-L^E397K Max Contact At (%) was significantly reduced compared to hNF-L Max Contact At (%) following recovery (D). The differences in means were analyzed for overall statistical differences using a two-way ANOVA with Holm-Sidak post hoc analysis. *, P < 0.05.

Figure 8. Limb coordination in hNF-L^E397K mice is altered after recovery from nerve injury

Perfectly coordinated diagonal (LH➔RF and RH➔LF) limbs have coupling values of ~0, whereas perfectly coordinated girdle (LH➔RH and LF➔RF) and ipsilateral (LH➔LF and RH➔RF) limbs have coupling values of ~50. Coordination failed to recover for all coupling values that included the left hind (LH) limb in hNF-L^E397K mice relative to pre-injury and hNF-L measurements (A, B and F). Coordination between limbs that did not include the left hind limb was unaffected following recovery (B, C and D). The differences in means were analyzed for overall statistical differences using a two-way ANOVA with Holm-Sidak post hoc analysis. *, P < 0.05.