Sialidase enhances spinal axon outgrowth in vivo

Abstract

The adult CNS is an inhibitory environment for axon outgrowth, severely limiting recovery from traumatic injury. This limitation is due, in part, to endogenous axon regeneration inhibitors (ARIs) that accumulate at CNS injury sites. ARIs include myelin-associated glycoprotein, Nogo, oligodendrocyte-myelin glycoprotein, and chondroitin sulfate proteoglycans (CSPGs). Some ARIs bind to specific receptors on the axon growth cone to halt outgrowth. Reversing or blocking the actions of ARIs may promote recovery after CNS injury. We report that treatment with sialidase, an enzyme that cleaves one class of axonal receptors for myelin-associated glycoprotein, enhances spinal axon outgrowth into implanted peripheral nerve grafts in a rat model of brachial plexus avulsion, a traumatic injury in which nerve roots are torn from the spinal cord. Repair using peripheral nerve grafts is a promising restorative surgical treatment in humans, although functional improvement remains limited. To model brachial plexus avulsion in the rat, C8 nerve roots were cut flush to the spinal cord and a peroneal nerve graft was inserted into the lateral spinal cord at the lesion site. Infusion of Clostridium perfringens sialidase to the injury site markedly increased the number of spinal axons that grew into the graft (2.6-fold). Chondroitinase ABC, an enzyme that cleaves a different ARI (CSPGs), also enhanced axon outgrowth in this model. In contrast, phosphatidylinositol-specific phospholipase C, which cleaves oligodendrocyte-myelin glycoprotein and Nogo receptors, was without benefit. Molecular therapies targeting sialoglycoconjugates and CSPGs may aid functional recovery after brachial plexus avulsion or other nervous system injuries and diseases.

Fig. 1.

Rat model of brachial plexus nerve avulsion injury with peripheral nerve graft. (A) Surgical preparation before closure. The spinal cord in the region of C8 is revealed, with the peroneal nerve graft extending from its insertion site in the ventrolateral aspect of the spinal cord toward its coaptation with the suprascapular nerve (not visible). A catheter extending from an osmotic pump is anchored, by a suture, to the dura just caudal to the graft insertion site. (B) Fixed (demonstration) preparation. After perfusion–fixation of the rat, the spinal cord, peroneal nerve graft, and coapted suprascapular nerve were dissected, allowing visualization of the bridging graft. In experiments using retrograde tracer (data not shown), 4 weeks after implantation the peroneal nerve graft was recut 7 mm distal to its spinal cord insertion site and sealed in a microreservoir of Fluoro-ruby dye.

Fig. 2.

Spinal neurons retrogradely labeled via a peroneal nerve graft. Horizontal sections of the spinal cord are shown in the area surrounding the peripheral nerve graft. The graft is visible as a roughly circular cross section containing labeled spinal axons, surrounded by retrogradely labeled spinal neurons. Red fluorescent images (indicating Fluoro-ruby retrograde staining) are presented as reverse grayscale for clarity. (A–F) Control (saline)-treated animals display some retrogradely labeled neurons (A). Animals treated with PI-PLC (B) (20 units/ml) appear similar to controls, whereas sialidase (C and D) (0.38 units/ml) and chondroitinase ABC (E and F) (0.5 units/ml) display significantly greater numbers of stained axons and spinal neurons. Most of the retrogradely labeled neurons are adjacent to the graft (but see Fig. 4). (Scale bar, 200 μm.)

Fig. 3.

Quantification of retrogradely labeled spinal neurons in control and enzyme-treated animals. Average total retrograde-labeled neurons in spinal cord sections from animals treated with saline (Control, n = 12), chondroitinase ABC (ChABC, n = 11), PI-PLC (n = 6 at each concentration), and sialidase (n = 6 at each concentration) are shown (mean ± SEM). Treatment with chondroitinase ABC (0.5 units/ml) and sialidase (0.38 units/ml) each resulted in significantly greater peripheral nerve graft innervation compared with the saline-treated control (∗, P ≤ 0.005 by Student’s t test).

Fig. 4.

Innervation of peripheral nerve grafts by remote neurons. Although most of the retrogradely labeled neurons are adjacent to the graft (see Fig. 2), it was not uncommon to find more distant labeled neurons near the central canal or on the contralateral side (A and B, arrows; B Inset). In some sections, the continuity of axons was traced from distant labeled spinal neurons to the graft (A and B, color-enhanced). Some axons spanned multiple segments in the horizontal sections (C, arrowheads). Treatments were: 0.38 units/ml sialidase (A) and 0.5 units/ml chondroitinase ABC (B and C). (Scale bar, 200 μm.) [Scale bar (Inset), 50 μm.]