Cellular Delivery of Neurotrophin-3 Promotes Corticospinal Axonal Growth and Partial Functional Recovery after Spinal Cord Injury

Lesion surgery

T7 dorsal laminectomies were performed on rats deeply anesthetized with a mixture (2 ml/kg) of ketamine (25 mg/ml), rompun (1.3 mg/ml), and acepromazine (0.25 mg/ml). The dura was opened, and limited dorsal column (n = 8) or more extensive bilateral dorsal hemisection lesions (n = 7) were performed using a fine-tipped glass-pulled aspiration device (Tuszynski et al., 1996). To make dorsal column lesions, the dorsal cord midline was identified and superficially incised with microscissors. The aspiration device, with a 22 ga core diameter, was then used to extend the lesion laterally to the lateral edges of the dorsal columns and ventrally to the level of the CST where it lies just dorsal to the central gray matter and central spinal canal. The CST was then aspirated fully at the T7 level; the transition from CST to dorsal portion of the central gray matter was readily identifiable as a distinct color change from white to gray matter. The last corticospinal fibers conspicuously adhered to the aspiration device tip and literally lifted away from the central gray matter, marking complete interruption of corticospinal fibers and arrival to a point immediately dorsal to the mid-dorsoventral axis of the cord. The aspiration procedure was extended slightly more ventrally and laterally to ensure resection of all dorsal CST axons. Lesion extent was verified by complete interruption of anterograde transport of WGA-HRP injected into the hindlimb sensorimotor cortex (see below) and by examination of serial Nissl-stained sections. To perform dorsal hemisection lesions, the dorsal columns and dorsal CST were removed as indicated above. Using the dorsal column/corticospinal lesion as a guide for the desired dorsoventral depth of the lesion, the lesion was extended laterally to remove the lateral aspects of the cord bilaterally. After the operation, animals were kept warm, placed on beds of sawdust, and given manual bladder evacuation for a period of ∼10 d and intramuscular-ampicillin (25 mg twice per day) to prevent and treat urinary tract infections. Animals regained automatic neurogenic bladder function after 5–10 d.

Functional testing

Functional testing began 1 month after surgical lesions were placed and was repeated 2 months after surgery. Only healthy animals were included in functional analyses. Functional testing was based on methods reported by Goldberger et al. (1990) and Kunkel-Bagden et al. (1993). Results in lesioned animals were compared with findings in eight intact animals.

Grid locomotion (wire grid task). Animals were required to navigate across a 150 cm plastic grid runway containing 40 × 40 mm holes to reach a food reward, after food deprivation for 48 hr (no more than 10–15% loss of body weight). After 5 d of pretraining on the grid, subjects underwent 5 more days of testing, four trials per day. Footfalls (failure to grasp a rung resulting in drop of the foot below the plane of the grid) made while crossing the grid on the last day of testing were quantified using video monitoring. Data were presented as number of footfalls to cross the platform averaged across the four trials on the final day of testing.

Platform locomotion with footprint analysis. Animals were placed on an 8-cm-wide × 8-foot-long platform with a food reward at the end. During 5 d of training on the runway, rats learned to walk toward the food reward, thereby producing continuous locomotion. Animals were tested for 5 additional days after the hind paws were inked (left, blue; right, red), and they ambulated on white paper. Each footprint consisted of the paired footprint pads with five toe prints. A total of 10 footprint pairs were examined from the final day of testing, using sets of footprints containing at least three consecutive strides. The following measurements were made: (1) stride length, the distance between foot pads on two consecutive footprints; (2) base of support, distance between right and left foot; and (3) angle of rotation, the angle of intersection between lines defined by the angle of the footpad and toes, drawn according to standardized criteria (Kunkel-Bagden et al., 1993). Ten samples from each subject were analyzed, and individual subject means were determined.

Elevated platform task. The forelimbs of the rats were placed on a Velcro pad of a platform located 18 inches above ground level. The latency to climb onto the top of the platform was measured. Intact subjects normally climb onto the platform with ∼1 sec latency, using their hindlimbs to assist the climb onto the platform. After 5 d of pretesting (three trials/day), latencies were quantified from three additional trials per day conducted over a 3 d period. Results from the nine total trials were averaged and compared. Comparisons between groups were made using ANOVA with post hoc Fisher’s least square difference.

Lesion completeness was verified by anterograde tracing of the CST and Nissl staining at the conclusion of functional testing. For anterograde tracing of the corticospinal projection, 300 nl of a 4% solution of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) (Sigma, St. Louis, MO) was injected through pulled-glass micropipettes (40 μm internal diameter) into each of 12 sites spanning the rostrocaudal extent of the rat sensorimotor cortex (Paxinos and Watson, 1986) using a PicoSpritzer II (General Valve, Fairfield, NJ). Air-driven pulses of 15 nl per pulse, 20 pulses per site were delivered with a 2 sec latency between pulses. The micropipette tip remained in place for 30 sec before withdrawal. Animals were transcardially perfused 48 hr later with 1% paraformaldehyde/1.25% glutaraldehyde followed by 10% buffered sucrose. Thirty-five-micrometer-thick sections were cut in the sagittal plane and divided into a series of six sections. Three of every six sections were reacted with a modification of the tetramethyl benzidine (TMB) method of Mesulam (1978), and the remaining sections were Nissl-stained. TMB-reacted sections were viewed using a dark-field condenser attached to an Olympus BM-1 microscope. Lesion completeness in animals that underwent dorsal column lesions was verified by complete interruption of WGA-HRP transport in TMB-reacted sections and by loss of all dorsal column white matter on Nissl-stained sections. Lesion completeness in animals that underwent dorsal hemisection lesions was similarly determined by loss of all WGA-HRP transport and by loss of dorsal spinal cord white and gray matter on Nissl-stained sections visualized in the coronal plane.

Anterograde labeling of the CST projection and quantification of CST growth after injury

Methods used to inject WGA-HRP are described in Experiment 1. To measure the amount of CST growth in lesioned subjects, WGA-HRP granules were quantified using National Institutes of Health (NIH) Image software. Measurements were controlled for differences in efficiency of WGA-HRP labeling between animals (see below). Labeling was performed in 10 NT-3-grafted animals and 15 control-lesioned animals by quantifying the density of WGA-HRP reaction product under dark-field illumination at the level of the lesioned CST (0 mm), and 4, 8, and 12 mm distal to the most caudal aspect of the lesioned CST. Any labeling artifact was edited out of sections before quantification. The number of pixels occupied by reaction product in every labeled section was quantified using NIH Image software on a video image of each 100× magnified section transmitted by a high-resolution Sony CCD camera. Thresholding values were chosen that maximized contrast between reaction product and background and were held constant between all subjects. A fixed box size of 552 × 436 pixels at 100× magnification corresponding to a sample area of 0.24 mm² was used to sample each subject. Total labeled pixels at each distance from the lesioned CST were quantified in every HRP-labeled section (every 9 of 10 sagittal-sectioned sections per animal) to generate the total density of sprouted CST fibers at each distance from the transected CST. To correct for differences in HRP labeling efficiency between animals, a baseline labeling density measurement (BLDM) was established. To determine the BLDM, the density of the labeled CST was measured at a point 1.5 cm rostral to the lesion site in each subject. Then the pixel values at each level (0, 4, 8, and 12 mm distal to corticospinal lesion) were divided by the animal’s own BLDM compensation factor. These corrected values were then compared between animals to determine a specific and direct measurement of CST growth. The presence of significant differences in growth among groups was determined by ANOVA.

Immunolabeling

Animals were transcardially perfused with 100 ml of cold 0.1m PBS followed by 300 ml of 4% paraformaldehyde in PBS. Spinal cords were removed, post-fixed overnight in 4% paraformaldehyde in PBS, and then left for 3 d in phosphate buffer (PB) containing 30% sucrose at 4°C. Sagittal sections were cut at 35 μm intervals with a cryostat. Every sixth section was immediately mounted on glass slides for Nissl staining. Remaining alternate sections were processed for immunocytochemical labels for the low-affinity p75 NGF receptor (monoclonal IgG-192 antibody at 1:100 dilution; gift of Dr. C. E. Chandler, Department of Neurobiology, Stanford University), neurofilament (NF) [RT97 monoclonal antibody from Boehringer Mannheim (Mannheim, Germany) against 200 kDa NF at 1:250 dilution)], choline acetyltransferase (ChAT) (for cholinergic fibers; polyclonal rabbit antibody at 1:5000 dilution; gift of Dr. L. G. Hersh, Department of Biochemistry, University of Kentucky), tyrosine hydroxylase (TH) (for dopaminergic and noradrenergic fibers; monoclonal antibody from Incstar at 1:1000 dilution), dopamine β hydroxylase (DBH) (for noradrenergic fibers; polyclonal rabbit antibody from Eugene Tech at 1:3000 dilution), serotonin (5-HT) (polyclonal rabbit antibody from Eugene Tech at 1:1000 dilution), calcitonin gene-related peptide (CGRP) [for sensory fibers; polyclonal rabbit antibody from Chemicon (Temecula, CA) at 1:8000 dilution] (Skofitsch and Jacobwitz, 1985; Harmann et al., 1988; McNeill et al., 1991), and glial fibrillary acidic protein (GFAP) (monoclonal antibody from Boehringer Mannheim at 1:250 dilution). All immunocytochemical labeling was performed by (1) incubating free-floating sections for 24 hr in primary antibody solution in 0.1m Tris-saline containing 1% blocking serum and 0.25% Triton X-100; (2) incubation for 1 hr with biotinylated goat anti-rabbit IgG (for polyclonal antibodies) or biotinylated horse anti-mouse IgG (for monoclonal antibodies; Vector Laboratories, Burlingame, CA) diluted 1:200 with Tris-saline containing 1% blocking serum; (3) 1 hr incubation with avidin–biotinylated peroxidase complex (Vector Elite Kit) diluted 1:1000 with Tris-saline containing 1% blocking serum; and (4) treatment for 3–15 min with 0.05% solution of 3.3′ diaminobenzidine, 0.01% H₂O₂, and 0.04% nickel chloride in 0.1 m Tris buffer. Immunolabeled tissue sections were mounted onto gelatin-coated glass slides, air-dried, dehydrated, and covered with Permount and glass coverslips. Sections were examined microscopically for graft survival and lesion extent. Immunolabeled sections were examined to determine the phenotype and extent of fiber penetration within grafts.

Double-label immunofluorescence confocal microscopy

To identify the nature of host/graft interactions that might influence axonal penetration into the intraspinal cell grafts, double-labeling for NF and GFAP was performed in four animals (two NT-3 and two uninfected fibroblast control grafts). Subjects were perfused with 4% paraformaldehyde in 0.1 m PB. Spinal cords were removed and serially sectioned in the sagittal plane on a cryostat at 35 μm intervals. Sections were rinsed 3× 10 min each in TBS (0.1m). Sections were incubated in TBS + 5% normal horse serum + 0.25% Triton-X for 1 hr and then transferred into the first primary antibody directed against NFs [monoclonal RT97 antibody at 1:175 dilution (Boehringer Mannheim)] and incubated overnight at 4°C on a rotating platform. The following day, sections were rinsed 3× 10 min each in TBS + 0.25% Triton-X and then incubated in horse anti-mouse biotinylated secondary (1:200; Jackson Immunochemicals, West Grove, PA) for 2.5 hr. Sections were then rinsed 3× in TBS and incubated in dichlorotriazinylamino fluorescein (DTAF)-streptavidin as a tertiary (1:300; Jackson Immunochemicals) for an additional 2.5 hr. Sections were rinsed again in TBS and then blocked in TBS + 5% normal donkey serum for 1 hr. Sections were then incubated in the second primary, GFAP (polyclonal, 1:750; DAKO, Carpinteria, CA), overnight at 4°C. The following day, sections were incubated in secondary donkey anti-rabbit-Texas Red (1:200; Jackson Immunochemicals) for 2.5 hr and then rinsed. Double-immunolabeled sections were mounted onto glass slides and coverslipped with FluoroMount-G (Southern Biotechnology, Alabaster, AL) and observed using absorption spectra filters of bandpass 490 (DTAF) and 545 (rhodamine). Sections were observed and imaged using a Bio-Rad MRC-1024 confocal microscope (Bio-Rad, Richmond, CA) to examine the association of reactive glial processes to neuritic patterns of labeling.

In vivo transgene expression

The ability of grafts of human NT-3-expressing cells to maintain transgene expression over time in vivo was assessed in separate animals by performing RT-PCR on fresh dissections of NT-3-producing cell grafts to nonlesioned spinal cords. RNA was isolated from fresh cord using the method of Chomczynski and Sacchi (1987). One microgram of total RNA was reverse-transcribed according to manufacturer’s instructions (Boehringer Mannheim) using random primers. The 50 μl PCR reaction contained 1/10 of first-strand synthesis, 0.5 μg of each primer, 1.5 mmMgCl₂, 50 mm KCl, 10 mmTris-HCl, pH 9.0, 0.1% Triton X-100, 0.2 mm dNTP, and 2.5 U Taq polymerase (Promega, Madison, WI), and amplification was performed for 35 cycles (60 sec at 94°; 30 sec at 60°C; 60 sec at 72°C). Sequences of primers are published elsewhere (Senut et al., 1995). Ten microliters of each PCR reaction were separated in a 2% agarose gel. Grafts in two animals each were tested at time points of 2 weeks, 1 month, and 3 months, and two additional animals were sampled at 6 months to gauge the extent of prolonged in vivotransgene expression.

Statistics

Differences in quantitative variables between groups were tested by ANOVA. Post hoc differences were assessed by Fisher’s least significant difference. For all data collection, experimenters were blinded to group identities.

Function

On functional testing, recipients of NT-3-secreting grafts showed significant recovery on the grid task compared with uninfected fibroblast control graft recipients (Fig. 3) at 1 and 3 months after grafting (p < 0.01). Recipients of NGF-secreting grafts did not show functional recovery (Fig. 3), indicating the specificity of the functional effect to recipients of NT-3-secreting cell grafts. NT-3 graft recipients performed significantly better than the other lesioned groups on the grid task but also differed significantly from intact animals. As observed in the first set of experiments above, deficits on platform motor tasks that did not require extensive sensorimotor integration were not detected, nor did NT-3 grafts impair these functions (Fig. 4). Similarly, deficits were not present on the elevated platform task (data not shown).

Histology

Findings were investigated in all NT-3- and control-grafted animals [findings in NGF-grafted animals have been reported previously and are not repeated here (Tuszynski et al., 1994, 1996, 1997)]. Grafts of NT-3-secreting and control uninfected fibroblasts survived in the lesion cavity through the 3 month grafting period (Fig.5). WGA-HRP labeling of the injured corticospinal projection demonstrated significant growth of CST axons in recipients of NT-3-secreting grafts at and distal to the spinal cord lesion site compared with control animals (Figs. 6,7). Growth was significant for up to 8 mm distal to the lesion site. A statistically significant increase in growth beyond this point, at 12 mm, was not observed. Of note, corticospinal axons extended through spinal cord gray matter but not into white matter tracts. Furthermore, only the lesioned dorsal CST appeared to extend axons in response to the presence of the NT-3-secreting graft, whereas an enhancement in sprouting of the unlesionedventral CST was not observed. The latter finding was evident in two ways. First, WGA-HRP-labeled axons of the ventral CST were not observed to traverse the ventral white to gray matter interface, whereas numerous axons crossed the interface between the dorsal CST and gray matter. Second, the number of WGA-HRP-labeled axons in the ventral CST did not differ between NT-3-grafted and control uninfected fibroblast-grafted subjects: 5.9 ± 1.5 axons per section were labeled in the ventral CST in NT-3-grafted subjects compared with 5.1 ± 1.2 axons per section in control-lesioned subjects (p = 0.83). Thus, axons of thelesioned dorsal CST rather than axons of the intact ventral CST responded to NT-3-secreting grafts, and contributions of the CST to functional recovery, if any, were likely derived from the dorsal rather than ventral CST. In some cases, WGA-HRP labeling revealed distinct growth from the tips of lesioned CST axons at the injury site to points distal, representing regeneration of injured axons. In other cases, WGA-HRP labeling that occurred at and distal to the injury site was punctate in nature (Fig. 6) and could represent either regeneration or sprouting of axons near the injury site.

Axonal/glial associations

In no case did corticospinal axons penetrate NT-3-secreting or control grafts. This lack of CST penetration into grafts could result either from glial/inflammatory responses at the host/graft interface that blocked growing axons or from components of the graft substrate that were nonpermissive for axon growth. To determine the association of spinal cord axons with glia at the lesion site and at the host/graft interface, sections double-labeled for NF and GFAP were examined. At the host/graft interface, significant upregulation of GFAP expression was observed (Fig. 8); however, several NF-labeled processes readily penetrated regions of GFAP upregulation to pass through the glial “barrier” and directly penetrate both NT-3-secreting and control grafts. Indeed, both NF- and GFAP-IR processes continued to penetrate grafts for some distance, and NF- and GFAP-IR processes were often co-associated within grafts (Fig. 8). In other instances, NF-IR processes penetrated grafts without specific co-association with GFAP-IR processes. The overall nature of the astroglial response did not differ between NT-3 and control graft recipients. Thus, glial responses at the host/graft interface did not present an impenetrable wall to lesioned axons; on the contrary, in some instances astrocytic processes were intimately co-associated with penetrating axons.

Responses of other axonal phenotypes to NT-3-secreting cell grafts

Immunolabeling revealed penetration of some axonal phenotypes into both NT-3-secreting and control grafts but no significant augmentation of this growth in NT-3-secreting graft recipients (Fig.9). NF immunolabeling revealed modest axonal growth into both graft types (Fig. 9a,b). Specific labeling to identify the transmitter phenotype of penetrating axons indicated that most originated from primary sensory afferents, evidenced by immunolabeling for CGRP (Fig. 9c,d) and Substance P, with occasional responses from 5-HT- (Fig. 9e,f), TH- (Fig.9g,h), and DBH-labeled axons. No significant sprouting responses were observed from ChAT-immunolabeled axons into either NT-3 or control grafts. Alterations in immunolabeling patterns of these markers in host regions surrounding the graft were not observed, distinct from the specific augmentation in growth of WGA-HRP-labeled CST axons in NT-3 graft recipients (Figs. 6, 7). There were no differences in axonal growth within the host cord when lesioned nongrafted animals and lesioned fibroblast-grafted animals were compared.

In vivo expression of the human NT-3 transgene for the 3 month period of this experiment, and indeed for at least 6 monthsin vivo, was verified by RT-PCR performed on fresh grafts placed in unlesioned rat spinal cords (Fig. 10). Because these measurements were made in grafts to the unlesioned cord, the possibility exists that expression might have differed in grafts to lesioned spinal cords.