Sialidase enhances recovery from spinal cord contusion injury

Abstract

Axons fail to regenerate in the injured spinal cord, limiting motor and autonomic recovery and contributing to long-term morbidity. Endogenous inhibitors, including those on residual myelin, contribute to regeneration failure. One inhibitor, myelin-associated glycoprotein (MAG), binds to sialoglycans and other receptors on axons. MAG inhibition of axon outgrowth in some neurons is reversed by treatment with sialidase, an enzyme that hydrolyzes sialic acids and eliminates MAG-sialoglycan binding. We delivered recombinant sialidase intrathecally to rats following a spinal cord contusive injury. Sialidase (or saline solution) was infused to the injury site continuously for 2 wk and then motor behavior, autonomic physiology, and anatomic outcomes were determined 3 wk later. Sialidase treatment significantly enhanced hindlimb motor function, improved bulbospinally mediated autonomic reflexes, and increased axon sprouting. These findings validate sialoglycans as therapeutic targets and sialidase as a candidate therapy for spinal cord injury.

Fig. 1.

Sialidase efficacy in vivo. Rats were fitted with intrathecal catheters to deliver carrier (saline solution containing 1 mg/mL rat serum albumin) or sialidase (2 U/mL in carrier) to the T10 level of the spinal cord, and then were subjected to spinal cord contusion injury at T9. An initial dose of 50 μL was delivered via the catheter, followed by infusion of the same solution to the site via osmotic pump (0.5 μL/h). After 12 d, rats were perfused with fixative agent, spinal cords were dissected, and horizontal cryosections were prepared and immunostained with anti-GT1b monoclonal antibody (A–C) or anti-GM1 monoclonal antibody (D–F) and fluorescent secondary antibody. Composite microscopic images encompassing the injury site and adjacent spinal cord are shown. (A and D) Spinal cord from a saline solution-infused (control) rat demonstrates intense anti-GT1b antibody immunostaining (A) and little GM1 immunostaining (D); (B and E) A section from the saline solution-treated (control) rat was overlaid with sialidase in vitro (2 U/mL, ambient temperature, 16 h) before immunostaining, resulting in elimination of anti-GT1b antibody immunostaining (B) and appearance of the sialidase product, GM1 (E). (C and F) Spinal cord from a sialidase-infused rat demonstrating loss of anti-GT1b immunostaining throughout the spinal cord (C) and appearance of anti-GM1 immunostaining (F). (Scale bar: 1 mm.)

Fig. 2.

Sialidase enhances recovery of hindlimb locomotor function after spinal cord injury. After spinal cord contusion injury, rats received an intrathecal bolus of sialidase or carrier, and then were infused with the same solution via osmotic pump (0.5 μL/h) for 2 wk. Hindlimb motor function was quantified by using the BBB scale periodically for 35 d after injury. (A) Average BBB scores (mean ± SEM) as a function of time after injury for control (n = 11) and sialidase-treated (n = 14) rats. Both groups display the same partial recovery (BBB score, 11) during the first 2 wk, and then diverge over the last 3 wk, with sialidase treatment resulting in significantly enhanced hindlimb function. *P < 0.05. (B) BBB scores at 35 d after injury for each rat in the study. (C) BBB improvement (mean ± SEM) over the period 2 to 5 wk after injury; *P < 0.02.

Fig. 3.

Sialidase enhances spinal-mediated autonomic function after spinal cord injury. Contused rats received intrathecal sialidase or saline solution (control) as described in the text. At 5 to 6 wk postinjury, rats were anesthetized, the renal sympathetic nerve was surgically isolated, and its activity recorded in response to drug-induced blood pressure fluctuations (Fig. S1). (A) Average normalized RSNA response range (mean ± SEM) for saline solution–treated (control, n = 6) and sialidase-treated (n = 7) rats; *P < 0.05. (B) RSNA responses for each rat in the study.

Fig. 4.

Sialidase treatment increases serotonergic axons caudal to a spinal cord contusion injury. After spinal cord contusion injury, rats received intrathecal delivery of sialidase or saline solution (control) as described in the text. At 35 d after injury, rats were perfusion-fixed and their spinal cords dissected. Transverse sections 7 mm caudal to the lesion were immunostained for serotonergic fibers and immunoreactivity quantified in the ventral horns. (A and B) 5-HT–immunostained ventral horns from control (A) and sialidase-treated (B) rat spinal cord sections. (C and D) Boxed areas in A and B, respectively, enlarged to show the characteristic “beads on a string” appearance of 5-HT stained axons. (Scale bar: 200 μm.) (E) Average 5-HT–immunopositive pixel areas (mean ± SEM) for saline solution–treated (control, n = 6) and sialidase-treated (n = 6) rats; *P < 0.05.