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.

The injured CNS is highly inhibitory for axon regeneration, severely limiting functional recovery (1). This results in part from axon regeneration inhibitors (ARIs) that accumulate at injury sites, including molecules on residual myelin [i.e., myelin-associated glycoprotein (MAG), NogoA, and oligodendrocyte-myelin glycoprotein) and chondroitin sulfate proteoglycans on astrocytes in the glial scar (2–5). ARIs bind to complementary receptors on axons to signal outgrowth arrest. Blocking ARI–receptor interactions or ARI signaling pathways may improve axon regeneration or sprouting and enhance recovery from CNS injury. Experimental therapies to address this goal include competitive inhibitors of ARI–axon binding, inhibitors of ARI-induced signaling pathways, and treatments that destroy or block the biosynthesis of ARIs or their receptors (4, 6–9). One such treatment, delivery of the bacterial enzyme chondroitinase ABC to remove chondroitin sulfate chains from proteoglycans in the injured spinal cord, shows promise in preclinical trials (10, 11). The current study extends the enzyme therapy approach, using sialidase to remove sialic acids from neural tissue and reverse MAG–sialoglycan binding.

MAG binds to various neuronal receptors to inhibit axon outgrowth, including Nogo receptors (NgR1 and NgR2), β1-integrin, PirB, and sialoglycans (gangliosides GD1a and GT1b) (12). Some neurons respond to MAG primarily via sialoglycans, whereas others use NgRs and other receptors (13, 14). Gangliosides are the most abundant sialoglycans on nerve cells (15). In some nerve cells, MAG-mediated axon outgrowth inhibition in vitro is reversed by blocking ganglioside biosynthesis, by competitive inhibition using MAG-binding sialoglycans (16), and by sialidase, an enzyme that cleaves the key MAG-binding terminal sialic acid from GD1a and GT1b (14, 17).

Sialidases (also called neuraminidases) are expressed across the phylogenetic spectrum (18). Some bacterial sialidases are readily overexpressed as recombinant proteins, are highly stable, and efficiently cleave sialic acid residues from MAG-binding sialoglycans on living neurons without cytotoxicity. Previously, we found that infusion of a recombinant sialidase to the site of a peripheral nerve graft enhanced outgrowth of CNS axons into the graft (19). Here, we report that sialidase enhances motor and autonomic functional recovery and stimulates axon outgrowth after spinal cord contusion injury in the rat, an animal model of the most common type of spinal cord injury in humans (20).

Results

Sialidase Production, Stability, and in Vivo Enzyme Efficacy.

Recombinant Vibrio cholerae sialidase was expressed in Escherichia coli at high yield (>500 U/L culture) and was purified chromatographically to very high purity (>100 U/mg protein) (21). Sialidase was highly stable as formulated for intrathecal delivery; analysis of sialidase activity recovered from implanted catheters after 12 d in vivo revealed retention of 90 ± 9% (mean ± SEM) of enzyme activity.

To test sialidase as a treatment to improve recovery after spinal cord contusion, rats were assigned to one of two groups: carrier (saline solution containing 1 mg/mL rat serum albumin) or sialidase (2 U/mL in carrier). Treatments were coded, and evaluators were blinded to the treatment group. Rats were fitted with an intrathecal catheter threaded to T10, and then a moderate contusion (175 kdyn) was delivered to the exposed spinal cord at T9 using a force sensor feedback-controlled Infinite Horizon impactor. Carrier or sialidase was delivered via the catheter immediately after the injury as a bolus (50 μL), then continuously via osmotic pump (0.5 μL/h) for the ensuing 2 wk. In a limited pharmacokinetic study in acutely treated animals, the bolus injection of sialidase resulted in a rapid increase up to approximately 1 U/mL in cerebrospinal fluid recovered from T9. Over the following 6 h of delivery by osmotic pump, sialidase in the cerebral spinal fluid equilibrated at 30 to 60 mU/mL

Sialidase delivered intrathecally over the course of treatment effectively cleaved sialic acid residues from spinal cord sialoglycans (Fig. 1). Efficacy was evaluated using highly specific monoclonal antibodies to gangliosides GT1b and GM1 (22). The trisialoganglioside GT1b, a major brain sialoglycan and a receptor for MAG, is expressed intensely in the gray matter and less intensely in the white matter of the spinal cord (Fig. 1A). Treatment of control spinal cord sections in vitro with sialidase eliminated anti-GT1b immunostaining (Fig. 1B). Similarly, intrathecal infusion of sialidase in vivo for 12 d resulted in nearly complete loss of GT1b (Fig. 1C), with only small foci of punctate staining remaining. GT1b was depleted over entire longitudinal sections extending up to 10 mm caudal and 4 mm rostral to the contusion. Intrathecally delivered sialidase penetrated throughout the spinal cord; deep longitudinal sections showed equivalent GT1b loss from the pia to the central canal. The product of sialidase action on GT1b is the monosialoganglioside GM1, whose internally located sialic acid is resistant to V. cholerae sialidase. Before sialidase treatment, GM1 expression was low and restricted to white matter tracts (Fig. 1D). Sialidase treatment in vitro or in vivo resulted in robust increases in GM1 staining in the gray matter wherever GT1b was depleted (Fig. 1 E and F).

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.)

Sialidase-Mediated Improvement of Motor Behavior.

Hindlimb function was evaluated in control and sialidase-treated contused rats by investigators blind to the treatment group using the Basso, Beattie, Bresnahan (BBB) 21-point (0, paraplegia; 21, normal) locomotor rating scale (23). All rats retained in the study displayed severe hindlimb paralysis during the first 4 d after impact (BBB score ≤ 4). Characteristic of moderate contusion injury in the rat, all animals spontaneously recovered partial hindlimb function within 2 wk of injury, reaching an average score of approximately 11 regardless of treatment group (Fig. 2A). It is notable that the course of partial recovery in control and treatment groups was identical over the 2 wk following the injury, as restorative processes would not be expected to be manifested over this short time period. Notably, at 5 wk after injury, control rats remained near this level (average, 12.6 ± 0.7), whereas sialidase-treated rats recovered significantly greater hindlimb function (15.6 ± 1.1; P < 0.05). One half of the sialidase-treated group but fewer than 10% of control rats reached a BBB score of at least 16, indicative of consistent coordination and frequent toe clearance (Fig. 2B). As spontaneous recovery was equal for control and sialidase-treated groups during the first 2 wk, we calculated hindlimb functional improvement individually for each rat over the final 3 wk of the trial (Fig. 2C). On average, sialidase-treated rats increased their hindlimb score by five points on the BBB scale compared with an average improvement of less than two points for control rats (P < 0.02).

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.

Sialidase-Mediated Improvement of Autonomic Function.

Spinal cord injury often results in autonomic dysreflexia, including fluctuations in blood pressure that add significantly to long-term morbidity (24). Autonomic control of blood pressure is mediated, in part, by pathways that project from the brainstem to the spinal cord and that regulate activity of sympathetic nerves, including renal sympathetic nerve activity (RSNA) (25). Most of the sympathetic preganglionic neurons that generate RSNA are located between T10 and L1 (26, 27).

Normally, an increase in blood pressure results in a compensatory decrease in RSNA, and vice versa. This was simulated in rats using drugs to modulate blood pressure and measuring the resultant changes in RSNA (Fig. S1). Spinal cord contusion injury resulted in a diminished range of RSNA responsiveness (57% of the predrug baseline; Fig. 3A). Notably, treatment with sialidase robustly enhanced responsiveness, doubling the response range to 114% of baseline (P < 0.05). The majority (>70%) of sialidase-treated rats, but fewer than 20% of control rats, attained a response range of greater than 100% of baseline (Fig. 3B).

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.

Sialidase Did Not Reduce Contusive Tissue Damage.

Spinal cord injury is a complex event; initial trauma is followed by neuroinflammation and secondary tissue damage (28). As sialoglycans mediate inflammation (29), we asked whether sialidase treatment decreased tissue damage. Longitudinal spinal cord sections were stained with eriochrome and cresyl violet to label myelin and cell bodies and volumes of white matter, gray matter, lesioned tissue, and lesion cavity were calculated. Sialidase treatment did not spare white or gray matter, nor did it reduce the size of the lesion or cavity (Fig. S2). We conclude that sialidase treatment improved recovery without limiting tissue damage after spinal cord contusion injury.

Sialidase-Mediated Enhancement of Axon Sprouting.

Therapies that reverse ARIs may increase axon sprouting and outgrowth generally, or may selectively affect nerve subpopulations (13, 14). In the current study, anterograde labeling of corticospinal tract (CST) axons, for example, did not reveal a significant treatment-related increase in labeled axons within 7 mm caudal (P = 0.7) or rostral (P = 0.8) to the lesion. These data are consistent with other studies in which behavioral improvements in rodents were seen in the absence of CST regeneration (30, 31).

In contrast, serotonergic [i.e., 5-hydroxytryptamine (5-HT)] axon density was increased caudal to the injury site in sialidase-treated animals (Fig. 4). Serotonergic fibers were detected immunohistochemically in transverse sections 7 mm caudal to the lesion center (Fig. 4 A–D). Quantitative image analysis (Fig. 4E) revealed a 22% increase in 5-HT–positive fibers in the ventral horns of sialidase-treated rats compared with control-treated rats (P < 0.05). As descending 5-HT projections from the brainstem to ventral horn motoneurons modulate locomotor reflexes (32), these findings are consistent with the conclusion that sialidase-mediated enhancement of axon regeneration, axon sparing, or axon sprouting improved functional recovery.

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.

Discussion

This preclinical study demonstrates that intrathecal delivery of sialidase to the site of a spinal cord contusion injury results in enhanced motor and autonomic function and increased axon sprouting. Although motor improvement was variable, half the sialidase-treated rats recovered significant hindlimb function. Sialidase treatment also resulted in a substantially greater range in baroreceptor-mediated renal sympathetic nerve activity (greater than twice the average of control-treated rats) in five of seven animals tested. Although the number of animals in the current study was limited, enhanced functional outcomes in blinded evaluations were statistically significant.

The enzyme used in these studies, V. cholerae sialidase overproduced in E. coli (21, 33), has advantages as a potential biological drug. It is produced at high concentration (>500 U/L) in bacterial culture, is readily purified, and is remarkably stable, with little loss of activity even after 12 d in vivo. Furthermore, V. cholerae sialidase robustly removes cell surface sialic acids from living neurons in culture without toxicity, reversing MAG-mediated axon outgrowth inhibition in some nerve cell types (17, 34). Intrathecal infusion of 2 U/mL of V. cholerae sialidase in the current studies did not result in toxicity and effectively cleared sialoglycans from a large area of spinal cord tissue surrounding the infusion site.

V. cholerae sialidase removes terminal sialic acids from complex gangliosides, converting them to the simpler ganglioside GM1. Conversion of GT1b to GM1 was confirmed in vivo after infusion of sialidase to the spinal cord (Fig. 1). It is reasonable to propose that the therapeutic benefit of sialidase was a result of conversion of MAG-binding gangliosides (GD1a and GT1b) to GM1, which does not bind MAG (35). Supporting this hypothesis are data demonstrating that sialidase reverses the inhibitory effects of MAG on axon outgrowth from some types of neurons in vitro (13, 14, 17). The increase in serotonergic fibers caudal to the lesion in the current study and our prior observation that sialidase enhanced axon outgrowth from motor neurons into a peripheral nerve graft (19) provide evidence that sialidase treatment enhances axon outgrowth in vivo. However, V. cholerae sialidase has broad specificity for sialic acids in various linkages to lipids and proteins (21). Sialic acid in its various forms, linked as monomers or polymers, has a multitude of direct and indirect effects on cell surfaces, the modification of which may impact axon regeneration (36). Furthermore, GM1, which is increased by sialidase treatment (Fig. 1), may have axon-promoting properties (37, 38). Additional studies are required to identify which sialidase substrates and/or products are most responsible for improved functional recovery. Regardless of which cellular and molecular mechanisms are responsible, the present study establishes the feasibility of producing and delivering sialidase to the contused spinal cord, and its potential for therapeutic benefit.

Materials and Methods

Sialidase.

V. cholerae sialidase was overexpressed in E. coli using an expression plasmid [pET30b(+)/VCNA] provided by G. Taylor (Fife, Scotland, UK) (21). Sialidase activity was determined using the fluorogenic substrate 4-methylumbelliferyl-N-acetylneuraminic acid (Sigma-Aldrich) (39), with 1 U defined as 1 μmol 4-methylumbelliferone released per minute at pH 7.3. Expression and purification were essentially as described (21). Overexpressed protein was extracted from E. coli using Bugbuster 10× Protein Extraction Reagent (EMD) diluted in 10 mM Tris HCl, 2.5 mM NaCl, pH 7.6. Cell debris was removed by centrifugation (8,000 × g, 30 min) and the enzyme was purified from the supernatant by sequential ammonium sulfate precipitation, anion exchange chromatography (HiTrap Q HP; GE Healthcare), and size exclusion chromatography (Sephacryl S-200 HR; GE Healthcare) as described (21). Active fractions from size exclusion chromatography were pooled and resubjected to anion exchange chromatography, eluting with a stepwise salt gradient. Active fractions were pooled and dialyzed against 20 mM Tris-HCl, 100 mM NaCl.

Purified sialidase was formulated for infusion by dilution (>50-fold) to a final concentration of 2 U/mL in a saline carrier solution containing 137 mM NaCl, 2.7 mM KCl, 6.4 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 0.5 mM CaCl₂, and 1 mg/mL rat serum albumin (Sigma-Aldrich). Control rats received carrier solution without sialidase.

Spinal Cord Contusion and Sialidase Infusion.

All procedures were approved by the Johns Hopkins Animal Care and Use Committee consistent with federal law and National Institutes of Health regulations. Johns Hopkins Medical Institutions are accredited by the American Association for Accreditation of Laboratory Animal Care. Female Sprague-Dawley rats (230–250 g) were anesthetized with an i.p. mixture of ketamine (60 mg/kg; Phoenix Pharmaceutical) and medetomidine (40 mg/kg; Pfizer). The dorsal skin was shaved and sterilized; a dorsal thoracic midline incision was made between T8 and L1 and paraspinal muscle removed between T9 and T13. After laminectomy at T13, an intrathecal catheter (PE-60 tubing heated and stretched to an external diameter of approximately 0.25 mm) was inserted subdurally and threaded rostrally to T10. Silk sutures placed between L1 and L2 were used to anchor the catheter in place. A laminectomy was performed at T9 to accommodate the tip of an Infinite Horizon impactor (Precision Systems). The spine was stabilized with clamps at the T8 and T10 vertebrae, and a 175-kdyn contusion was delivered at T9 (20). Sialidase solution (50 μL) or carrier was delivered to the injury site via the intrathecal catheter and the proximal end of the catheter was connected to an s.c. osmotic pump (200 μL, 0.5 μL/h, 14 d; Alzet/Durect) prefilled with the same solution. After skin closure, animals were revived from anesthesia using atipamezole (0.25 mg per animal; Pfizer). Fluid supplementation (lactated Ringer solution) and gentamicin (5 mg/kg; Quality Biological) were administered daily for the first week after surgery. Bladder expression was performed twice daily for 1 wk and then daily for the following week after surgery or until bladder function returned.

Behavioral Testing.

On days 1, 4, and 7 after injury, and weekly thereafter, hindlimb motor function was assessed using the BBB open-field locomotor test (23). Behavioral analysis was performed by two observers blinded to the treatments. A small number of animals that received a score of 5 or higher at day 4 after injury were considered to have insufficient initial injury and were removed from the study.

Neurophysiology.

For acute baroreflex experiments, rats were initially anesthetized in a plastic chamber and then with a nose cone, using 2% isoflurane in O₂. Isoflurane was discontinued after administration of α-chloralose (100 mg/kg i.v.; Sigma) via a left jugular vein cannula. The jugular vein catheter was also used to administer gallamine triethiodide. The depth of anesthesia was maintained at a surgical plane by supplemental doses of α-chloralose (25 mg/kg). The depth of anesthesia was determined either by testing corneal reflexes before and during the recovery from paralysis, or by the variability of RSNA and arterial blood pressure (AP) when rats were paralyzed. Body temperature was monitored with a rectal probe and maintained at 35 °C to 37 °C with a heating pad and lamp. The trachea was cannulated for mechanical ventilation using a rodent ventilator (CWE). The right femoral artery was cannulated for measurement of AP. Arterial pressure and heart rate were recorded simultaneously with Micro1401 hardware and Spike 2 software (Cambridge Electronic Design). The left and right femoral veins were cannulated for the separate administration of depressor and pressor drugs.

Preparation for RSNA recording has been described (40). The left kidney was approached via a left flank laparotomy and retracted. The adrenal gland, the fat covering the psoas muscle, and the paraspinal muscles were deflected from the renal nerve, which typically was located at the junction of the aorta and the renal artery or was found traversing the aorta. The renal nerve was dissected from the renal vasculature and surrounding tissue with the aid of an operating microscope. The renal nerve was then immersed in mineral oil and mounted on a bipolar hook electrode connected to a differential amplifier with a bandpass of 300 to 3,000 Hz. Sympathetic activity was further processed by rectification and low-pass filtering at a time constant of 0.1 s and recorded continuously with the arterial pressure and heart rate.

Baroreflex function curves (Fig. S1) were obtained by plotting the reflex change in RSNA to increases and decreases in AP caused by the vasodilator sodium nitroprusside (SNP, 50 μg/mL) and the α-adrenergic agonist phenylephrine (PE, 125 μg/mL), respectively, in successive ramped infusions. In intact rats, AP is sensed by receptors in the carotid arteries and aorta. The activity of these receptors is processed in the brainstem, and pathways descending from the brainstem to the spinal cord modulate sympathetic activity inversely with respect to AP. This modulation is disrupted or eliminated caudal to spinal cord lesions [most of the sympathetic preganglionic neurons that generate RSNA are located between T10 and L1 (26, 27)].

SNP was administered first, beginning at a rate of 2.5 mL/h and increased by 2.5 mL/h every 30 s until an AP of 60 mm Hg below baseline or a maximum rate of 25 mL/h was reached. Following SNP administration, PE was immediately administered beginning at a rate of 2.5 mL/h and increasing by 2.5 mL/h every 30 s These infusions produced an approximately linear increase in AP from 60 mm Hg below baseline AP to 40 mm Hg above baseline AP at a rate of approximately 1.5 mm Hg/s. The RSNA was analyzed from the SNP-induced nadir (60 mm Hg below baseline) in AP to the PE-induced peak AP (40 mm Hg above baseline), as shown in Fig. S1A. Baroreflex function, the average RSNA as a function of blood pressure, was fit to a sigmoidal curve (41), and the response range (ΔRSNA) was determined (Fig. S1D).

CST Tracing.

Four weeks after injury, rats were anesthetized with a mixture of ketamine (15 mg/kg) and medetomidine (10 mg/kg) and the skull above the sensorimotor cortex was removed. Biotinylated dextran amine (100 mg/mL in water; Molecular Probes) was injected (1 μL/injection) unilaterally at eight points in the sensorimotor cortex. Animals were revived from anesthesia with atipamezole (0.25 mg per animal) and maintained for 2 wk before perfusion.

Perfusion and Tissue Processing.

At 5 or 6 wk after injury, animals were anesthetized and transcardially perfused with 4% paraformaldehyde in Dulbecco PBS solution (42). Spinal cords were dissected and postfixed 12 to 16 h in the same fixative agent, then cryoprotected in 30% sucrose. The spinal cord was embedded in Shandon M-1 Embedding Matrix (Thermo Scientific) and serial 15 μm cryosections were cut in sets on a longitudinal plane and thaw-mounted onto Super-Frost Plus slides (Thermo Scientific). Transverse cryosections of spinal cord were collected 7 to 8 mm rostral and caudal to the injury site.

Histology and Immunohistochemistry.

For histochemical analysis of the lesion area, longitudinal sections were rehydrated and stained with eriochrome and cresyl violet (43), dried, then coverslipped with DPX mounting medium (Electron Microscopy Sciences). For immunohistochemistry, slides were pretreated for 2 h at ambient temperature with a solution of 5% donkey serum, 1% BSA, and 0.5% Triton X-100 in PBS solution, then were incubated in antibody against serotonin (5-HT, 1:5,000; ImmunoStar) overnight at 4 °C. After three washes in PBS solution, sections were incubated with Cy2-conjugated donkey antirabbit antibody (1:200, 2 h, ambient temperature; Jackson ImmunoResearch). Sections were washed and coverslipped with Krystalon (EMD Chemicals). For visualization of biotinylated dextran amine–labeled fibers, slides were incubated with Cy3-streptavidin (1:200, 2 h, ambient temperature; Jackson ImmunoResearch), washed, and coverslipped as described earlier.

Microscopic images were analyzed by observers blind to the treatment. For analysis of lesion volume, a 3.5 mm × 3.5 mm box was centered on the lesion cavity of eriochrome/cresyl violet–stained slides and areas of white matter, gray matter, lesion tissue, and cavity were outlined in adjacent sections. Volumes were calculated using Stereo Investigator software (MBF Bioscience). Anterograde-labeled CST fibers were counted in longitudinal sections at 1-mm increments rostral and caudal to the lesion epicenter. Fluorescently labeled serotonergic fibers were quantified in a boxed region over the ventral horns of three transverse sections 7 mm caudal to the lesion. Positive fibers were distinguished from background staining and quantified using NIS-Elements software (Nikon). Relative pixel areas represented by 5-HT were converted into total fiber areas.

Sialidase Efficacy in Vivo.

Rats were fit with catheters and contused (200 kdyn) as described earlier. Twelve days after injury and treatment, rats receiving sialidase or saline solution (control) were perfusion-fixed, their spinal cords dissected and cryoprotected, and 15-μm longitudinal sections collected onto slides. Some fixed sections from control rats were treated in vitro with sialidase before staining by overlaying them with a solution containing 2 U/mL sialidase, incubating at ambient temperature for 16 h, then washing with PBS solution. Sections from control rats (treated and untreated in vitro) and from sialidase-treated rats were blocked by incubating in PBS containing 1% BSA and 5% goat serum for 5 h at 4 °C, and then were incubated in the same buffer containing anti-GT1b or anti-GM1 monoclonal antibody (mAb's GT1b-1 or GM1-1, 1 μg/mL) (22) for 16 h at 4 °C. Slides were washed with PBS solution and then incubated in the same buffer containing Cy3-labeled goat anti-mouse IgG (7 μg/mL; Jackson ImmunoResearch) for 16 h at 4 °C. Sections were washed with PBS solution and water, dried, and mounted as described earlier. Composite fluorescent images were acquired and compiled using a Nikon Eclipse 90i microscope.

Statistics.

Comparisons between sialidase- and control-treated groups were performed via an unpaired t test.