The Armadillo as a Model for Leprosy Nerve Function Impairment: Preventative and Therapeutic Interventions

Abstract

Mycobacterium leprae infection of peripheral nerves and the subsequent nerve function impairment (NFI), especially in response to reactional episodes, are hallmarks of leprosy. Improved treatments for M. leprae-induced nerve injury are needed, as most if not all of the disability and stigma associated with leprosy arises from the direct or indirect effects of NFI. Nine-banded armadillos (Dasypus novemcinctus), like humans, exhibit the full clinical spectrum of leprosy and extensive involvement of the peripheral nerves. In this study, state-of-the-art technology was used to compare nerve function between uninfected and M. leprae-infected armadillos. Motor nerve conduction velocity (MNCV) and compound muscle action potential (cMAP), which measure changes in the rate of impulse conduction velocity and amplitude, revealed a progression of impairment that was directly correlated with the duration of M. leprae infection and enabled development of an objective nerve impairment scoring system. Ultrasonography accompanied by color Doppler imaging detected enlargement of the M. leprae-infected nerves and increased vascularity, possibly due to inflammation. Assessment of epidermal nerve fiber density (ENFD), which shows a length-dependent innervation in armadillos that is similar to humans, identified small fiber degeneration early after M. leprae infection. Staining for neuromuscular junction (NMJ) integrity, which is an indicator of signal transduction efficiency into skeletal muscle, discerned a markedly lower number and structural integrity of NMJ in M. leprae-infected armadillo footpads. These tools for assessing nerve injury were used to monitor the effects of intervention therapy. Two potential neuro-protective drugs, ethoxyquin (EQ) and 4-aminopyridine (4-AP), were tested for their ability to ameliorate peripheral nerve injury in M. leprae-infected armadillos. 4-AP treatment improved MNCV, cMAP, and EFND compared to untreated animals, while EQ had less effect. These results support the armadillo as a model for M. leprae-induced peripheral nerve injury that can provide insights toward the understanding of NFI progression and contribute to the preclinical investigation of the safety and efficacy of neuro-preventive and neuro-therapeutic interventions for leprosy.

Keywords: Dasypus novemcinctus; Mycobacterium leprae; armadillos; leprosy; nerve function impairment; neuropathies.

FIGURE 1

Localization of M. leprae in the posterior tibial nerve of infected armadillos. (A) Posterior tibial nerve showing clumps of intraneural M. leprae (arrows); modified Fite/Faraco stain. Electron micrographs showing M. leprae (M. lep) within unmyelinated (B) and myelinated (C) Schwann cell (SC) located by the axon (AX) in the post-tibial nerve of an armadillo at 24 months post-experimental infection.

FIGURE 2

Posterior tibial nerve conduction wave forms in uninfected armadillos. Motor Nerve Conduction Tests (MNCT) were performed on the posterior tibial nerve of uninfected armadillos (N = 25) using a Cadwell Sierra Summit portable electrophysiology unit. Representative wave forms are shown of a right posterior tibial (A) and left posterior tibial (B) nerve. Measurements were taken from the proximal stimulation site to the active electrode and distal stimulation site to the active electrode to measure the distance between stimulation sites. Compound muscle action potential (cMAP) was measured from onset (O) to the peak (P) in both ankle and knee in response to supra-maximal electrical stimulation.

FIGURE 3

Posterior tibial nerve conduction wave forms showing reduced compound muscle action potential (cMAP) in M. leprae-infected armadillos. MNCT were performed on the posterior tibial nerves of M. leprae-infected armadillos (N = 71). Representative motor nerve conduction wave forms depict early (A) (4 months infection) and mid (B) (9 months infection) stage leprosy disease and show abnormal wave forms with declining degrees of cMAPs (<0.9 mV). Armadillos progressing to late stage leprosy disease (12–24 months post-inoculation), exhibit conduction block at the knee (C).

FIGURE 4

MNCT scores decrease with duration of infection and correlate with anti-PGL1 antibody titers. A longitudinal study (A) demonstrated negative correlation between the MNCT score and duration of M. leprae infection, indicating progressive nerve injury (N = 20). (B) Phenolic glycolipid I (PGL-1) antigen was used in an enzyme immunoassay to detect anti-PGL1 IgM circulating antibody levels in armadillo serum samples. Armadillos with high (>0.900 OD 540 nm) anti-PGL1 antibody titers have lower MNCT scores, while those with low (0.700–0.900 OD 540 nm) anti-PGL-1 antibody titers have higher MNCT scores (N = 71).

FIGURE 5

M. leprae infection causes nerve enlargement in armadillo peripheral nerves. Ultrasonography of the posterior tibial nerve was performed using a SmartUS transducer probe with a linear broadband frequency of 1.5–15 MHz connected to a Sierra Summit basic system. The posterior tibial nerve is shown along its longitudinal (A) and transverse (B) axes in an uninfected armadillo and a M. leprae-infected armadillo [(C,D), respectively]. The nerve diameter of the M. leprae-infected armadillo is 0.096 cm² indicating nerve enlargement compared to the nerve diameter of the uninfected armadillo (0.037 cm²). Ultrasound with color Doppler imaging in M. leprae-infected armadillos showed increased blood flow in animals at late stage leprosy disease (23 months post-infection) that were positive for anti-PGL-1 antibodies (E) compared to armadillos that were negative for anti-PGL-1 antibodies at 9 months post-infection (F). The intense colors indicate lower velocity of the blood flow traveling away and toward the transducer (N = 4).

FIGURE 6

M. leprae infection causes atrophy of armadillo footpad muscles. Photographs of the dissection of a representative animal showing (A) the small lateral flexor muscle of the footpad and how the fibers run between the tendons and (B) the lumbrical muscles of the right hind limb that were released from their tendon of insertion. (C) Atrophy of small lateral flexor and lateral lumbrical muscles in M. leprae-infected armadillos compared to uninfected armadillos. (D) Atrophy of medial and lateral insertions of the medial lumbrical muscle in M. leprae-infected armadillos (N = 10) compared to uninfected armadillos (N = 8). *p < 0.05.

FIGURE 7

M. leprae infection causes degeneration of neuromuscular junctions (NMJ’s) in the armadillo footpad muscles. The number of NMJs (A) is reduced (**p = 0.0012) in M. leprae-infected armadillos (terminal stage) as measured in 30μm-thick, 30² mm muscle sections. Fluorescent micrograph (B) of footpad intrinsic muscle in an M. leprae-infected armadillo showing NMJs (green, arrowheads) stained with FITC-α-bungarotoxin and muscle fibers (red) stained with PE-anti-myosin. (N = 8).

FIGURE 8

Evaluation of the effect of neuroprotective drugs on NFI in M. leprae infected armadillos. (a) Armadillos treated with 4-Aminopyridine- (4-AP) (N = 4) and Ethoxyquin (EQ) (N = 3) showed a significantly higher (***p = 0.001 and **p = 0.012, respectively) cMAP (mV) than the untreated controls (N = 5) at 1 month post-treatment. (b) Epithelial nerve fiber density (ENFD, #fibers/mm) in 4-AP- and EQ-treated and untreated armadillos. (c) Skin section from an untreated armadillo stained with PGP9.5 showing epidermal nerve fibers (arrows) terminating at the epidermis (epi). (d) Skin section from a 4-AP-treated armadillo showing more branching of the epidermal nerve fibers (arrows) and that the dermal nerve bundles are dense at the papillary dermis (derm) in comparison to the untreated armadillo. (e) Skin section from an EQ treated armadillo showing single epidermal nerve fibers (arrows) entering the epidermis. Scale bars = 50 μm.