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Abstract

The mechanisms by which neural precursor cells (NPCs) enhance functional recovery from spinal cord injury (SCI) remain unclear. Spinal cord injured rats were transplanted with wild-type mouse NPCs, shiverer NPCs unable to produce myelin, dead NPCs, or media. Most animals also received minocycline, cyclosporine, and perilesional infusion of trophins. Motor function was graded according to the BBB scale. H&E/LFB staining was used to assess gray and white matter, cyst, and lesional tissue. Mature oligodendrocytes and ED1⁺ inflammatory cells were quantitated. Confocal and electron microscopy were used to assess the relationship between the transplanted cells and axons. Pharmacotherapy and trophin infusion preserved gray matter, white matter, and oligodendrocytes. Trophin infusion also significantly increased cyst and lesional tissue volume as well as inflammatory infiltrate, and functional recovery was reduced. Animals transplanted with wild-type NPCs showed greatest functional recovery; animals transplanted with shiverer NPCs performed the worst. Wild-type NPCs remyelinated host axons. Shiverer NPCs ensheathed axons but did not produce MBP. These results suggest that remyelination by NPCs is an important contribution to functional recovery following SCI. Shiverer NPCs may prevent remyelination by endogenous cells capable of myelin formation. These findings suggest that remyelination is an important therapeutic target following SCI.

Keywords

Neural precursor cells (NPCs)Spinal cord injury (SCI)Transplantation Mechanism Remyelination

Introduction

Spinal cord injury (SCI) is uniquely devastating and leaves patients with deficits of motor, sensory, sexual, and sphincteric function. Clinicians are still without a safe and efficacious therapy to enhance recovery from SCI (21). Cellular transplantation is a promising new approach (47); however, little is currently known about how transplanted cells augment functional recovery or how exogenous cells interact with host tissue (52). Such insights will play a critical role in augmenting the modest functional recovery currently being seen with this approach and in ensuring its safety (21).

The goals of cellular transplantation strategies for SCI have been varied to date, as have the cells transplanted. Some have sought and achieved neuronal differentiation and integration into functional circuits (41,43,53). Others have sought to replace oligodendrocytes, remyelinating viable axons to restore their function (25,30,31,52). Nonneural cells such as bone marrow stromal cells have also shown benefit in the absence of neural differentiation or even long-term survival (16,34,42,65,67,70,71). A single beneficial mechanism such as trophin production could be common to all cells (8,10,34,52,64. Alternately, numerous cell type-specific mechanisms of similar efficacy are required to explain the results reported in the literature.

Our laboratory has established a combinatorial transplantation protocol whereby adult mouse brain-derived neural precursor cells (NPCs) are transplanted into the subacutely injured rat spinal cord (30). The present work aimed to confirm our group's previous findings as replication has been problematic in the regenerative medicine field (9). Moreover, it aimed to provide a detailed characterization of how the various therapeutics that have been administered have influenced functional recovery with the goal of establishing the beneficial mechanisms as this may lead to strategies to augment recovery. In particular, we sought to examine the importance of myelination to the mechanism of action of NPCs by using NPCs derived from MBP-null (shiverer) mice and comparing the effects with wild-type NPCs.

We provide evidence that remyelination by exogenous cells is an important mechanism leading to functional recovery in our protocol. Our data also suggest that remyelination by endogenous cells is of functional significance—which has not been widely accepted (20).

Materials and Methods

General Animal Care

All animal work was conducted in accordance with the Canadian Council of Animal Care guidelines and with institutional ethics approval (University Health Network, Toronto, Ontario, Canada). A total of 206 female, 10- to 12-week-old Wistar rats from Charles River Laboratories (Wilmington, MA, USA) were employed. Preoperative amoxicillin-clavulinic acid (Sigma-Aldrich, St. Louis, MO, USA) was routinely provided as infection prophylaxis, and buprenorphine (RB Pharmaceuticals Limited, Slough, Berkshire, UK) was administered for analgesia. Following injury, animals' bladders were manually evacuated three times per day until the return of normal voiding. Blinding was maintained for all assays.

Neural Precursor Cell Isolation and Culture

Adult mouse brain NPCs were harvested from the sub-ventricular zone of enhanced yellow fluorescent protein (eYFP) transgenic mice [strain 129-Tg(ACTB-EYFP) 2Nagy/J; Jackson Laboratory, Bar Harbor, ME, USA] in the fashion described previously (30,66). As a control for myelination, adult NPCs were also harvested from eYFP⁺shiverer mutant mice (Jackson Laboratory) bearing a deletion mutation in their myelin basic protein (MBP) gene (51). The shiverer mutation is recessive, and when homozygous, these mutant mice exhibit seizures, a characteristic tremor, and limited life span. To generate eYFP⁺shiverer mice, offspring from a cross of eYFP⁺ and shiverer mice were interbred. A stable line of homozygous eYFP⁺ shiverer mice was generated and employed as a source of NPCs that are unable to generate central nervous system myelin.

The mice described above were killed by cervical dislocation. Their brains were then excised under sterile conditions and transferred to sterile artificial cerebrospinal fluid (aCSF) solution containing 2 M NaCl, 1 M KCl, 1 M MgCl₂, 155 mM NaHCO₃, 108 mM CaCl₂, 1 M glucose, and 1% penicillin/streptomycin (all Sigma). The subventricular zone of the forebrain was dissected and transferred to a low-calcium aCSF solution (10 ml) containing 40 mg of trypsin, 20 mg of hyaluronidase, and 4 mg of kynurenic acid (all Sigma) for 30 min at 37°C. Following inactivation of trypsin, tissue was mechanically dissociated into a cell suspension with a pipette. Cells were plated on uncoated tissue culture flasks (Sarstedt, Montreal, Canada) in serum-free medium (200 ml) containing 20 ml of Dulbecco's modified Eagle's medium (DMEM)/F-12, 4 ml of 30% glucose, 3 ml of 7.5% NaHCO₃, 1 ml of 1 M 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 200 mg of transferrin, 50 mg of insulin, 19.25 mg of putrescine, 20 μl of selenium, 20 μl of progestrone, 1 μg of fibroblast growth factor 2 (FGF2), 2 μg of epidermal growth factor (EGF), and 1% penicillin/streptomycin (all from Sigma) for 7 days. The neurospheres generated were passaged weekly by mechanical dissociation in the same medium. Cells from the third passage (P3) were transplanted.

Experimental Protocol and Groups

The experimental protocol and group assignments are illustrated in Figure 1. Animals underwent moderate clip compression (SharpTech, Oakville, Canada) SCI (50) (closing force maintained 21.0 −23.0 g) for 1 min at the T7 vertebral level following a T6–T8 laminectomy. Two weeks later, animals were randomly assigned to experimental groups. With the exception of the Injured No Treatment group, all animals underwent a second surgery 2 weeks following their first. NPC + Growth Factor (GF)/Immunosuppression (IS) animals were trans planted with 4 × 10⁵ live wild-type eYFP⁺ NPCs. Shiverer NPCs + GF/IS animals were transplanted with 4 × 10⁵ live NPCs isolated from eYFP⁺shiverer mice bearing a deletion of the MBP gene (51). To control for the immune response to foreign organic matter, the Dead Cell + GF/IS group was transplanted with the same number of wildtype NPCs killed by 10 freeze–thaw cycles (40). Injured + GF/IS animals underwent sham transplantation with cell suspension media.

Figure 1.

Experimental protocol and groups. “GF” denotes growth factor administration via an implanted Alzet osmotic minipump at the time of cellular or control transplantation. Pharmacotherapy with minocycline and cyclosporine is denoted “IS” (immunosuppressants) here and throughout this manuscript. NPC: neural precursor cell; EGF: epidermal growth factor; FGF: fibroblast growth factor; PDGF: platelet-derived growth factor; eYFP: enhanced yellow fluorescent protein.

Cell and control transplantation was conducted at four intraspinal sites, approximately 2 mm rostral and caudal to the injury epicenter on both sides of the midline using a pulled glass needle (Sutter Instrument Company, Novato, CA, USA) was attached to a 5-μl Hamilton syringe (Reno, NV, USA) (62). Triturated cell suspension (2 μl) was aspirated prior to each injection, which employed a micromanipulator. A Micro 4 Microsyringe Pump Controller system (World Precision Instruments, Sarasota, FL, USA) was employed to provide an even injection rate over 5 min.

At the same surgical procedure, animals denoted “GF” were implanted with an osmotic minipump (Alzet model 1007D, primed overnight; DURECT Corporation, Cupertino, CA, USA) delivering a 100-μl mixture of EGF (3 μg/100 μl; Sigma), FGF (3 μg/100 μl; Sigma), and platelet-derived growth factor (PDGF; 1 μg/100 μl; Sigma) for 7 days. Distinct from our group's previous work, the intrathecal catheter tip was placed 1 cm rostral to the epicenter to reduce the risk of dislodgement. Animals denoted “IS” received a 10-day course of minocycline (50 mg/kg; Sigma; administered intraperitoneally) and daily cyclosporine (10 mg/kg; Novartis, Basel, Switzerland; administered subcutaneously) both starting 2 days prior to control or cell transplantation. Both drugs have well-known immunosuppressive and neuroprotective properties (12,19,32,38,73. Animals were sacrificed 1, 2, or 6 weeks following transplantation (3, 4, or 8 weeks following injury), depending on the intended investigations.

Functional Testing

All animals underwent open field motor scoring using the Basso, Beattie, and Breshnahan (BBB) scale weekly (6), beginning 1 week after injury by two experienced and blinded observers. Catwalk^© and gridwalk testing were planned, but too few animals recovered sufficient hindlimb motor function to justify these tests.

Histology and Immunohistochemistry

Tissue designated for histological analysis was preserved by means of transcardiac perfusion with 4% paraformaldehyde (Sigma). Following postfixation, tissue was embedded in OCT (Sakura Finetek USA, Torrance, CA, USA) and stored at −80°C. Upon sectioning, tissue was cut in 30-μm-thick axial slices using a cryostat (Leica Biosystems Inc., Concord, Canada) and then returned to storage at −80°C. Tissue was cut in sets to facilitate staining equivalent cord regions from each animal with hematoxylin and eosin/luxol fast blue (H&E/LFB; Sigma), anti-mast cell chymase mouse (CC1) for mature oligodendrocytes (1:40, Calbiochem, Darmstadt, Germany), ED-1 (antibody to rodent homologue of CD68) for activated microglia and macrophages (1:100, Serotec, Raleigh, NC, USA), and glial fibrillary acidic protein (GFAP) for astrogliosis (1:1,000, Chemicon, Temecula, CA, USA). Another set of these slides was used to quantitate the number of surviving transplanted NPCs in relevant animals.

H&E/LFB staining was employed to delineate white matter, gray matter, cyst, and lesional tissue. Cyst was defined as any region within the total circumference that was devoid of tissue. Lesional tissue included any abnormal-appearing tissue such as demyelinated white matter, fibrous and glial scarring. Image J software (NIH, Bethesda, MD, USA) was employed to measure tissue areas every 360 μm over a 10-mm region centered on the epicenter. Volumes were calculated using the Cavalieri method. The epicenter was considered the section bearing least gray and white matter. For volumetric calculations, the epicenter was considered a 2-mm region centered on the epicenter. The transplant sites were considered regions 1–4 mm rostral and caudal to the epicenter spanning the intended transplant sites.

For immunohistochemical staining, primary antibodies were diluted in blocking solution [1% bovine serum albumin (BSA; Sigma), 5% nonfat milk (Bio-Rad, Hercules, CA, USA), and 0.3% Triton X-100 (Sigma)] and incubated overnight at 4°C. The following day secondary antibodies were applied (Alexa 568, 1:400, Molecular Probes, Eugene, OR, USA) at room temperature for 1 h. Slides were coverslipped with Mowiol containing 4′,6-diamidino-2-phenylindole (DAPI; Calbiochem). To double stain for axons [heavy neurofilament 200 kDa (NF200); 1:500; Sigma] and MBP (1:1,000; Sternberger Monoclonal, Covance Inc., Montreal, Canada), the slides were stained with mouse anti-MBP antibody and antimouse Alexa 568 first followed by rabbit anti-NF200 and anti-rabbit Alexa 647 (1:400, Molecular Probes).

Astrogliosis was assessed by fluorescence intensity following staining with an anti-GFAP antibody (1:1,000). Staining was performed in large batches containing an equal amount of tissue from each group to minimize effects related to staining variability. Equivalent sections from injured and healthy control tissue samples were stained in each batch to ensure consistent staining between batches. Every second section (a distance of 360 μm apart) was photographed at 5× magnification using a Leica DMR8 fluorescent microscope (Buffalo Grove, IL, USA) with StereoInvestigator software (MBF Bioscience, Williston, VT, USA) and consistent hardware and software settings. We determined that the employed exposure time was in the linear range for fluorescence measurements. Red fluorescence intensity was subsequently measured with Image J software and multiplied by the tissue area measured. Using these values, the area under the curve was calculated.

Confocal microscopic images were obtained with a Nikon Eclipse TE 2000-U confocal microscope (Melville, NY, USA). Images illustrating the relationship between eYFP-bearing transplanted NPCs, axons, and MBP were instead assessed with a Zeiss LSM 510 META confocal microscope (Thornwood, NY, USA). This relationship was also assessed using immunoelectron microscopy.

Animals subject to immunoelectron microscopy were transcardially perfused with 4% PFA and 0.15% glutaraldehyde in phosphate buffer (PB; both Sigma). Postfixation was then performed with 4% PFA in 0.1 MPB at 4°C. Spinal cord tissue was embedded in 3% agar (agarose A; Rose Scientific, Edmonton, Alberta, Canada). A vibratome (Leica Biosystems Inc.) was used to cut 100-μm free-floating sections. The sections were preincubated in 0.1% H₂O₂ (Sigma) in PBS for 15 min to quench endogenous peroxidase activity. Sections were then blocked in 5% milk, 1% BSA, and 0.05% Triton X-100 in PBS and incubated in rabbit anti-green fluorescent protein (GFP) antibody (1:100; Chemicon) overnight at 4°C. The sections were washed in PBS three times and incubated in anti-rabbit horse radish peroxidase (HRP) secondary antibody (1:200; Sigma) overnight at 4°C. The sections were postfixed in 0.1% glutaraldehyde and then incubated with 0.04% diaminobenzidine (DAB; Sigma) for 30 min, followed by a second incubation with 0.04% DAB plus 0.005% H₂O₂ for 15 min. The sections were then treated with 1% osmium tetroxide (Electro Medical Systems, Nyon, Switzerland) in 0.1 M PB overnight, dehydrated in graded ethanol, and embedded in Araldite502/ Embed-812 (Electron Microscopy Sciences, Hatfield, PA, USA). Thin plastic sections were cut on an ultratome (Leica Biosystems Inc.), counterstained with uranyl and lead citrate (Sigma), and examined with a transmission electron microscope (Hitachi 7000; Hitachi, Tokyo, Japan).

Western Blotting

One-centimeter perilesional spinal cord samples were homogenized in radioimmunoprecipitation assay (RIPA; Thermo Scientific, Rockford, IL, USA) buffer. The samples were vortexed, then spun down in a centrifuge at 15,300 × g and 4°C for 15 min. The supernatant was collected and transferred to fresh tubes. A Lowry assay (Sigma) was performed on each sample to determine its total protein content. Samples with equal protein content were prepared based on these measurements. The prepared samples were run on a separating 12% polyacrylamide gel and transferred to a nitrocellulose membrane (both Bio-Rad). Coomassie Blue (Sigma) staining was employed to verify the presence of protein. The membrane was blocked with a 5% milk solution to minimize nonspecific binding. Blotting with a primary antibodies for 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase; 1:200; Chemicon) and ionized calcium-binding adapter molecule 1 (Iba1, 1:250; Wako Pure Chemical Industries, Osaka, Japan) was performed as well as housekeeping proteins glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Sigma; G8795, 1:7,000) and β-actin (Chemicon; MAB1501R, 1:400). The primary antibodies were placed on the membrane and incubated overnight. After washing, the membranes were incubated in a horseradish peroxidase-conjugated secondary antibody diluted 1:2,000 (Sigma, A78 3682) in blocking solution for 1 h at room temperature. The membrane was developed and exposed on X-ray film using an ECL chemilluminescence kit (PerkinElmer Lifesciences Inc., Boston, MA, USA). Images were generated for densitometry measurements using a Fluor-S MultiImager (Bio-Rad, Hercules, CA, USA), and measurements were made using Quantity One software (Bio-Rad, version 4.2.1).

Fluoro-Gold ™ Retrograde Axonal Labeling

To label neurons crossing the clip compression injury, 8 weeks following injury distinct designated animals from each group underwent Fluoro-Gold™ retrograde labeling. To achieve this, the animals underwent spinal cord transection at the T13 bony level 8 weeks following initial injury. A blunt hook was passed beneath the residual laminae to ensure the transection was truly complete as inadvertent incomplete transection can otherwise occur (60). A 0.5 × 0.5 × 0.5 cm Gelfoam^© pledget (Ethicon, Somerville, NJ, USA) soaked in 4% Fluoro-Gold™ (Fluorochrome, LLC., Denver, CO, USA) was inserted at the site of transection after hemostasis was achieved. After an additional week, animals were perfused as described above. For these animals, brains were harvested in addition to the spinal cords. The brains underwent 40-μm-thick axial cryosectioning to facilitate counts of retrogradely labeled neuron cell bodies of the red, vestibular, raphe, and reticular nuclei. eYFP⁺ total cell counts were performed on spinal cords from these animals, which were cut in 30-μm-thick axial sections to ensure successful NPC transplantation in relevant animals.

Cell Counting

A Leica DMR8 microscope with Stereoinvestigator software was employed to provide unbiased estimates of total counts of CC1- and ED1-stained cells at 20× microscopy. Counting parameters were similar to those previously published (47). Forty 100 × 100 μm optical fractionator counting frames were employed for each section. For these counts the presence of a nucleus within the cell body was required. The counts were performed on seven sections per animal, including the epicenter, ±500 μm, ±1,000 μm, and ±2,000 μm. After an initial blinded count of CC1⁺ cells, spinal cords bearing transplanted cells were recounted to determine the number of CC1⁺, eYFP⁺, and double-positive cells. This served to determine the number of oligodendrocytes, which were of endogenous or exogenous origin, and to verify the accuracy of the initial CC1⁺ cell counts as well as the accuracy of the total eYFP⁺ cell counts described below. Scheaffer CE values were usually between 0.1 and 0.2.

Total counts of eYFP⁺ NPC-derived cells were performed under 40× magnification using a Nikon Eclipse E800 fluorescent microscope. In all specimens, cut sections were scrutinized every 360 μm from the approximately 5-cm-long spinal cord segments and were examined for the presence of eYFP-bearing NPCs, which were readily distinguishable from background fluorescence. Cell bodies were counted, and these counts underwent Abercrombie correction (1). Animals that were found to be unsuccessfully transplanted after examination of the entire cut length of cord (8.6%; no significant difference in rate among the groups) were excluded from further analysis, as our goal was to determine the effect of transplanted cells. eYFP⁺ cells were counted in the spinal cords of animals subject to both the histological and Fluoro-Gold™ analyses. The total counts and length of eYFP⁺ cell distribution from both cohorts were pooled for analysis.

The cell bodies of Fluoro-Gold™-labeled neurons in the brainstem were counted using the fluorescent imaging capabilities of the Nikon Eclipse TE 2000-U confocal microscope. Low-power microscopy (2.5×) facilitated distinction of the red, rubrospinal, raphespinal, and vestibulospinal nuclei.

Statistical Analysis

SAS 9.2 software (SAS Institute, Cary, NC, USA) was used for statistical analysis. Means from continuous data were analyzed with analysis of variance (ANOVA). Subsequent intergroup comparisons were made with Bonferroni adjustment for multiple comparisons. Poisson regression was employed for count data. Parametric statistics were employed for BBB values in accordance with published recommendations (55). Proportions were analyzed by chi-square analysis.

Results

Neural Precursor Cell Survival Decreases with Time and Is Independent of Trophin Infusion or Pharmacotherapy

Data related to NPC survival, differentiation, and distribution are presented in Figure 2. Animals included in our experiments had an average of 38,435 surviving eYFP⁺ cells 2 weeks following transplantation (9.6%) (Fig. 2F). This was reduced to an average of 15,381 cells (3.8%) 4 weeks later. Between 2 and 6 weeks posttransplant, there was also a significant reduction in cell density (Fig. 2H). There was no evidence of eYFP⁺ cells in Dead Cell + GF/IS animals (data not shown).

Figure 2.

NPC survival, differentiation, and distribution in tissue following transplantation. (A) Transplanted eYFP⁺ (green) cells are shown in an axial spinal cord section 2 weeks following transplantation. Cell nuclei have been labeled blue with 4′,6-diamidino-2-phenylindole (DAPI). “V” denotes the ventral surface of the spinal cord. (B) Differentiation of a subset of eYFP-bearing NPCs into mast cell chymase mouse-positive (CC1⁺) mature oligodendrocytes is demonstrated. The lower visual field is a subset of the upper. (C) Distribution of NPCs is shown. For NPC + IS Only n = 9, for NPC + GF/IS n = 6. (D) NPC distribution is shown. For Shiverer NPC + GF/IS n = 7, for NPC + IS Only n = 5, for NPC + GF/IS n = 11. For (C) and (D), the epicenter is at 0 μm and (+) values are rostral. Horizontal red lines denote intended transplant sites. (E) The proportion of eYFP⁺ cells differentiating into CC1⁺ mature oligodendrocytes is shown. For the 1 week NPC + GF/IS group 439 of 1,724 counted NPCs were CC1⁺. For the 2 weeks NPC + IS Only group 4,304 of 8,642 counted NPCs were CC1⁺ and for the 2 weeks NPC + GF/IS 1,532 of 3,437 NPCs were CC1⁺. For the 6 weeks Shiverer NPC + GF/IS group 996 of 2,542 counted NPCs were CC1⁺, for the 6 weeks NPC + IS Only group 402 of 922 counted NPCs were CC1⁺, and for the 2 weeks NPC + GF/IS 1,107 of 2,436 NPCs were CC1⁺. (F) Average total cell counts are plotted for experimental groups at the 2 and 6 weeks time points. Pooled counts at 6 weeks are significantly less than those at 2 weeks (p < 0.0001). (G) Average lengths of NPC distribution are plotted for experimental groups at the 2 and 6 weeks time points. At the 6 weeks time point, length of distribution was significantly greater in the NPC + IS Only group as compared with the Shiverer NPC + GF/IS or NPC + GF/IS groups (p < 0.0001). (H) Average neural precursor cell density is plotted for experimental groups at the 2 and 6 weeks time points. Pooled densities at the 6 weeks time point are significantly lower than at the 2 weeks time point (p = 0.0416). For (F–H) n = 9 for the 2 weeks NPC + IS Only group and n = 6 for the 2 weeks NPC + GF/IS group. At the 6 weeks time point, n = 13 for the Shiverer NPC + GF/IS group, n = 10 for the NPC + IS Only group, and n = 20 for NPC + GF/IS group. Error bars represent standard error. ANOVA was employed to analyze means of continuous data. Subsequent intergroup comparisons were made using Bonferroni correction for multiple comparisons. Count data were analyzed using Poisson regression.

Plots of eYFP⁺ NPC-derived cell distribution relative to the epicenter noted that transplanted cells were predominantly localized nearer the epicenter than the transplant sites 2 mm rostral and caudal at 2 weeks posttransplant (Fig. 2C). The distribution of eYFP⁺ cells 4 weeks later was unchanged in animals implanted with a trophin pump; however, in the group that did not receive a trophin pump, NPCs were predominantly observed 3 mm rostral and caudal to the epicenter suggesting migration (Fig. 2D). Spread of the transplanted cells (distance between the most rostral and caudal eYFP⁺ cells in the sagittal plane) was less in animals implanted with a trophin pump at 6 weeks posttransplant (Fig. 2G). At both 2 and 6 weeks posttransplant, many cells appeared in cyst walls near the intended transplant sites, which are likely persisting injection cavities.

Figure 2E demonstrates that 1 week following transplantation 25.5% of transplanted eYFP⁺ NPCs had colabeled with CC1⁺ mature oligodendrocytes, demonstrating differentiation into mature oligodendrocytes. This increased to 48.3% by 2 weeks postinjury. This is similar to previously published observations from our group (30).

Trophin Infusion and Pharmacotherapy Are Associated with Gray Matter, White Matter, and Oligodendrocyte Sparing

By 6 weeks posttransplant pharmacotherapy and trophin infusion were associated with a significant increase in white matter volume (Fig. 3, Supplemental Fig. 1, www.drfehlings.ca/CT_GWJH_2012.html). Trophin infusion was also associated with significantly more gray matter at the epicenter by 6 weeks posttransplant. Of importance, the presence of exogenous cells alone did not increase gray or white matter volume.

Figure 3.

Analysis of tissue volumes in experimental groups 6 weeks posttransplant. (A) Sample histological measurements are presented for a single hemotoxylin and eosin/luxol fast blue-stained section (left). “V” denotes the ventral surface of the spinal cord. The total circumference is outlined in red, gray matter is outlined in green, cyst is outlined in orange, and lesional tissue is shaded blue. White matter area is calculated by substracting gray matter, cyst, and lesional areas from the total circumference. At right, tissue volumes of gray matter, white matter, cyst, and lesional tissue are plotted at the 6 weeks posttransplant time point. Positive values denote rostral to the epicenter, and negative values denote caudal to the epicenter. The epicenter was considered to be a 2,000-μm region centered on the section with least preserved gray and white matter. The injection sites were considered to span +4,000 μm to +1,000 μm and −1,000 μm to −4,000 μm relative to the epicenter. n = 12 for Injured No Treatment, n = 7 for Injured + GF/IS, n = 6 for Dead Cell + GF/IS, n = 7 for Shiverer NPC + GF/IS, n = 5 for NPS + IS Only, n = 11 for NPC + GF/IS. Error bars represent standard error. ANOVA was employed to analyze means, and subsequent intergroup comparisons were made using Bonferroni correction for multiple comparisons.

CC1⁺ cell counts of mature oligodendrocytes provided very similar results to those for white matter distribution and volume discussed above (Fig. 4). Analysis of white and gray matter volumes as well as oligodendrocyte counts in the Injured No Treatment group demonstrates an approximate 30% loss between 2 and 6 weeks, as might be expected in the context of ongoing secondary injury. Additionally, groups transplanted with NPCs did not show augmented levels of white matter or oligodendrocytes at either time point. This finding is supported by Western blot data from distinct animals 1 week post-transplant (Supplemental Fig. 2, www.drfehlings.ca/CT_GWJH_2012.html)—a time when a large proportion of transplanted NPCs were demonstrated to be CC1⁺.

Figure 4.

Oligodendrocyte numbers are preserved by pharmacotherapy and trophin infusion but are not increased by NPC transplantation. (A) Total oligodendrocyte counts for experimental groups are shown 2 weeks (A) and 6 weeks (B) following transplantation. Growth factor infusion was associated with significantly greater oligodendrocyte numbers only at the site closest to the catheter tip at 2 weeks posttransplant. The rostral location of the trophin pump tip is denoted as GF (p = 0.02668). At 6 weeks posttransplantation both trophin infusion and pharmacotherapy were significantly associated with oligodendocyte preservation (p = 0.0014 and p = 0.0037, respectively). For A and B, positive values are rostral to the epicenter, and negative values are caudal. +2,000 μm and −2,000 μm correspond to the rostral and caudal transplant sites, respectively. Error bars represent standard error. Data were analyzed using Poisson regression.

Fluorogold™ retrograde labeling of axons crossing the injury did not find evidence that any treatment preserved these axons based on overall counts (Fig. 5). Likewise, there was no suggestion of such an effect on the reticulospinal, raphespinal, vestibulospinal, or rubrospinal tracts (Fig. 5C–F).

Figure 5.

Neural precursor cell transplantation is not associated with axonal preservation. (A) Brightfield and fluorescent microscopic images provide representative images of brainstem nuclei labeled retrogradely by Fluoro-Gold™. The nuclei highlighted in yellow (middle) are shown in higher power on the right. (B) Overall counts of Fluoro-Gold™-labeled nuclei (endogenous NPCs) are presented for experimental groups 6 weeks posttransplant. (C–F) The component nuclei counts included in (B) are shown, which included reticulospinal (C), raphespinal (D), vestibulospinal (E), and rubrospinal neurons (F). There were no significant differences for B–F. n = 5 for Injured No Treatment, n = 5 for Injured + GF/IS, n = 6 for Dead Cell + GF/IS, n = 6 for Shiverer NPC + GF/IS, n = 5 for NPC + IS Only, and n = 9 for NPC + GF/IS. The Shiverer NPC + GF/IS group had an average of 15,615 surviving transplanted NPCs (eYFP labeled), the NPC + IS Only group had an average of 16,782 surviving NPCs, and the NPC + GF/IS group had an average of 9,709 surviving NPCs—no significant differences were observed. Error bars represent standard error. Data were analyzed using Poisson regression.

Trophin Infusion Increases Cyst, Lesional Tissue, and Inflammatory Infiltrate

Trophin infusion via an implanted osmotic minipump was associated with increased cyst and lesional tissue volumes 2 and 6 weeks postinsertion (Fig. 3, Supplemental Fig. 1, www.drfehlings.ca/CT_GWJH_2012.html). There is a suggestion of increased lesional tissue nearer the rostrally located tip of the trophin infusion catheter.

There was no statistical evidence for a difference in astrogliosis among experimental groups 6 weeks posttransplant as measured with fluorescence intensity (Supplemental Fig. 3, www.drfehlings.ca/CT_GWJH_2012.html). This is concordant with the findings of Hooshmand et al. (25).

Counts of ED1⁺-activated microglia and macrophages performed 6 weeks posttransplant demonstrated that trophin infusion significantly increased chronic inflammatory infiltrate 6 weeks postinjury, particularly when transplanted NPCs were present (Fig. 6). Similar to the lesional tissue results reported above, greater inflammatory infiltrate was noted nearer the infusion catheter tip. Western blot analysis performed in distinct animals 1 week posttransplant provided highly concordant results (Supplemental Fig. 2, www.drfehlings.ca/CT_GWJH_2012.html). These results are very similar to the pattern of inflammatory mediator expression previously observed [interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α] (22).

Figure 6.

Trophin pump implantation increases inflammatory infiltrate. (A) An immunohistochemically stained sagittal spinal cord section from an animal 2 weeks following transplantation is shown. Red ED1⁺ (antibody to rodent homologue of CD68-positive) cells closely approximate transplanted eYFP-bearing NPCs. ED1⁺ cells surround regions of NPCs suggesting a granulomatous inflammatory response. NPCs are predominantly localized to cyst walls, which are likely persisting injection cavities. (B) ED1⁺ cell counts performed in experimental groups 6 weeks posttransplant are presented. The distal tip of the growth factor (GF) infusion catheter is located more than 2,000 μm rostral to the epicenter as denoted by the red arrow. There were significantly more ED1⁺ cells in the NPC + GF/IS group than the NPC + IS Only or Injured No Treatment groups (p < 0.0001 and p = 0.0008, respectively). Trophin infusion via an osmotic minipump was associated with a significantly greater number of inflammatory cells (p = 0.0018). Error bars represent standard error. ANOVA was employed to analyze means of continuous data. Subsequent intergroup comparisons were made using Bonferroni correction for multiple comparisons. Count data were analyzed using Poisson regression.

Functional Recovery Is Related to the Myelinating Ability of Transplanted Cells

Although many variables were examined in this experimentation, most had little or no influence on functional recovery. The myelinating ability of transplanted cells was an important variable increasing or decreasing hindlimb motor function in comparison to an untreated SCI (Fig. 7). Animals receiving NPC transplantation and pharmacotherapy alone achieved the highest level of functional recovery. Moreover, they were twice as likely to achieve the motor milestone of weight support as compared with untreated animals (Fig. 7B). Trophin infusion decreased functional recovery based on a comparison of NPC + GF/IS and NPC+ IS Only groups. Transplantation of shiverer NPCs reduced functional recovery as compared with all other groups including Injured No Treatment animals demonstrating harm. A complete reporting of statistics related to intergroup comparisons for BBB data is provided in Supplemental Table 1 (www.drfehlings.ca/CT_GWJH_2012.html).

Figure 7.

Functional recovery is predicted by myelinating ability of transplanted cells. (A) Average Basso Beattie and Breshnahan (BBB) scores for surviving animals from each experimental group are plotted. The timing of cell or control transplantation is denoted by the vertical red line. ANOVA noted a significant difference in average values among experimental groups at 3 weeks (p = 0.0158), 5 weeks (p = 0.0090), 6 weeks (p = 0.0402), 7 weeks (p = 0.0493), and 8 weeks (p = 0.0157) postinjury. (B) The proportion of animals recovering an ability to weight bear (BBB > 8) is plotted for experimental groups. Differences between groups for (B) are not statistically significant. For both (A) and (B), sample sizes were as follows: Injured No Treatment n = 20, Injured + GF/IS n = 16, Dead Cell + GF/IS n = 16, Shiverer NPC + GF/IS n = 20, NPC + IS Only n = 11, NPC + GF/IS n = 26. Error bars represent standard error. ANOVA was employed to analyze means of continuous data. Chi-square was used to analyze the proportions presented in (B). Detailed statistical analysis is presented in Supplemental Table 1 (www.drfehlings.ca/CT_GWJH_2012.html).

Confocal and immune electron microscopy support the fact that wild-type exogenous eYFP⁺ cells ensheath axons and produce myelin in the NPC + GF/IS group as has been previously characterized in detail by our group (30) (Fig. 8). Here we additionally demonstrate remyelination by transplanted cells in the NPC + IS Only group (Fig. 8B–G, Supplemental Video 1, www.drfehlings.ca/CT_GWJH_2012.html). As expected, eYFP⁺ cells bearing the shiverer mutation did not show evidence of MBP production, but shiverer NPCs also ensheathed axons (Fig. 8K–M). The abnormal pattern of ensheathment is similar to the electron microscopic images of the shiverer mouse spinal cord published by Kirkpatrick et al. (33).

Figure 8.

Transplanted cells ensheath axons. (A) I–III demonstrate confocal images of tissue from the NPC + GF/IS group transplanted with wild-type NPCs, while IV–V demonstrate tissue transplanted with shiverer NPCs. III is a subset of II shown at higher power. For I–III white arrows demonstrate sites suggestive of remyelination by transplanted cells. IV and V demonstrate that shiverer NPCs also closely approximate axons but that these axons are devoid of myelin basic protein (MBP) staining, though MBP staining is seen elsewhere in the images. (B–G) A single confocal image from the NPC + IS Only group is presented at 60× magnification. In (B) DAPI staining is shown in blue denoting nuclei. In (C) eYFP is shown in green denoting exogenous NPCs. In (D) MBP staining is shown in red denoting myelin. In (E) heavy neurofilament 200 kDa (NF200) staining is shown in gray denoting axons. The merged image is shown in (F). Here a white arrowhead denotes an axon myelinated by a transplanted NPC. This is further delineated in (G) where a merge of eYFP (green) and MBP (red) is presented along with z-plane slices through the site of remyelination. Z-stack images through this field of view are shown in Supplemental Video 1 (accessible at www.drfehlings.ca/CT_GWJH_2012.html). (H) A region of positive immunoperoxidase staining for green fluorescent protein (GFP) denotes the location of eYFP⁺ cells within a tissue section. Brightfield microscopy has been performed. (I) Wild-type eYFP⁺ myelin wraps are seen in axial section ensheathing a host axon in the NPC + GF/IS group. The region delineated by the red box is shown in (J) in higher power. (J) Immunoperoxidase reaction precipitant is clearly seen denoting eYFP⁺ labeling. (K) Shiverer eYFP⁺ myelin wraps are seen. The region denoted by the red box is seen at higher power in (L). (M) Three images of increasing magnification are shown of an axon in sagittal section. Abnormal GFP⁺ wraps from shiverer NPCs are seen. The myelin wraps formed by shiverer NPCs seen here appear like those demonstrated in the uninjured shiverer spinal cord by Kirkpatrick et al. (33).

Discussion

Establishing the mechanism by which transplanted cells enhance recovery from SCI is important for many reasons; however, few strong conclusions can be drawn from literature published to date. Many groups have characterized one or a small number of putative mechanisms without a thorough exploration of the beneficial and negative interactions between exogenous cell and host. Functional benefit associated with transplantation of nonneural cells suggests that an interaction leading to neuroprotection or repair in the host is a likely mechanism for these cells and possibly for others. Supporting this notion, cell and tissue sparing has been noted with transplantation of neural (5,45,53,56,61 and nonneural cells (28,46,57,58) as well as reduced cavity formation (44,74) and the promotion of axon regeneration (18) and plasticity in the host spinal cord (4). Indeed, our group has demonstrated that our NPCs express trophins in vitro (23) and in vivo following transplantation (22).

Several recent publications provide important information regarding the mechanism by which transplanted cells may augment recovery following SCI. In a model in which human central nervous system (CNS) stem cells were transplanted into nonobese diabetic-severe combined immunodeficient (NOD-scid) mice, Cummings et al. demonstrated that the transplanted cells differentiated into remyelinating oligodendrocytes and synapse-forming neurons (13). Moreover, functional recovery was lost when the cells were ablated with diphtheria toxin, suggesting that functional benefit was related to an integrative rather than a neuroprotective process. Subsequent work by this group examined lesion volume, tissue sparing, descending serotonergic host fiber sprouting, chondroitin sulfate proteoglycan deposition, glial scarring, and angiogenesis (25). No evidence of host modification was found resulting from human CNS stem cell transplantation. Yasuda et al. recently published the first report of shiverer cell transplantation as a control for myelination in a mouse model (72). The authors found diminished functional recovery when shiverer cells were transplanted, and although few confounding factors were explored, they concluded that remyelination was an important mechanism of functional recovery in their model.

Here were present a detailed characterization of our laboratory's established combinatorial therapeutic strategy for rodent SCI, which incorporates murine NPC transplantation. Our results suggest both beneficial and harmful mechanisms, and both may be targets for enhancing functional recovery associated with cellular transplantation. It also suggests that remyelination by endogenous and exogenous cells following SCI is an important contributor to functional recovery. Though these findings are likely model specific, they are consistent with the findings of Cummings et al., Hooshmand et al., and Yasuda et al. (13,25,72). The importance of remyelination in our model is additionally supported by our group's previous work, which demonstrated augmented myelination (30) and electrophysiology (15) associated with NPC transplantation. This previous work did not, however, manipulate the myelinating ability of transplanted cells and thus did not clearly establish the functional importance of myelination.

Neural Precursor Cell Survival Decreases with Time and Is Independent of Trophin Infusion or Pharmacotherapy

Our group found that no experimental therapy was associated with a change in NPC cell survival or differentiation (27) nor was the MBP deletion in transplanted shiverer NPCs. The infusion of EGF, bFGF, and PDGF via an osmotic minipump was intended to support NPC survival and differentiation into oligodendrocytes (30), but neither effect was seen in this study. The more remote placement of the catheter tip may have led to lower concentrations at the epicenter and could explain this and the lower NPC numbers seen here than in our previous publication (30), as may differences in surgical technique.

Trophin Infusion via an Osmotic Minipump Affects NPC Location

It is interesting to note that in our study migration of transplanted cells was relatively limited in the rostrocaudal plane at 6 weeks and that the length of distribution decreased with time in animals implanted with a trophin pump. This is similar to the findings of Luo et al., who found a decrease in the area occupied by transplanted cells over time (37). Lack of distal migration in animals implanted with a trophin pump could be a result of restricted movement from increased scarring associated with their implantation.

Trophins Do Not Contribute to Functional Recovery

The mere presence of transplanted cells did not enhance functional recovery, suggesting that characterized and uncharacterized trophins produced by transplanted cells or in response to transplanted cells (22,23) are unlikely to contribute to functional recovery. Previous work from our group noted marked trophin elevation in the NPC + GF/IS group [delineated in detail by Hawryluk et al. (22)]. This was, however, insufficient to augment functional recovery in these animals; the possibility of beneficial effects masked by the detrimental effects of the trophin pump cannot be excluded, however.

Neuroprotective Effects Were Not Associated with Functional Recovery

In our transplant paradigm, we found evidence of gray matter, white matter, and oligodendrocyte sparing, which was attributable to trophin infusion and pharmacotherapy with no additional effect attributable to exogenous cells. The trophin infusion appeared to have greater effect in the transplantation regions, while pharmacotherapy appeared more efficacious near the epicenter. Our findings are similar to the report of Hooshmand et al. (25), who did not find tissue sparing attributable to transplanted cells. It is interesting that the tissue sparing observed in our study was without functional recovery. It is possible that the lack of functional benefit stems from the adverse effects ascribed to the trophin pump to ongoing compression from the infusion catheter or to lack of concomitant axonal preservation. Indeed, we cannot exclude that the benefit in the NPC + IS Only group may have been related to neuroprotection without the harmful effects of a trophin pump rather than to myelination. Comparison of the shiverer NPC + GF/IS group to the NPC + IS Only and Injured No Treatment is problematic, as these groups differ by more than one variable. Direct comparison to the NPC + GF/IS, Dead Cell + GF/IS, and Injured + GF/IS groups is, however, facilitated by the fact that these groups differ only by a single variable. The detrimental functional effect seen following transplantation of shiverer NPCs suggests an important effect attributable to myelination. It is interesting to consider that the lack of benefit associated with neuroprotection seen here is similar to the results seen with neuroprotective agents in human clinical trials (24). In this context, it is possible that remyelination could be a more important therapeutic target.

It is also interesting to consider that myelination could also be an important mechanism of benefit for other transplanted cells. Several publications suggest that bone marrow stromal cells (BMSCs) or similar nonneural cells could enhance remyelination either by forming Schwann cell-like cells (11,14,36,68,75 or by stimulating endogenous remyelination (2,3,54). Future experimentation thoroughly characterizing the mechanism underlying the benefits of nonneural cell types will be very important for advancing this field.

Myelin Basic Protein Deletion Alters the Inflammatory Response to Neural Precursor Cells

It was unexpected that the deletion of the MBP gene would markedly alter the inflammatory response seen in the comparison between the NPC + GF/IS and shiverer NPC + GF/IS groups, which were otherwise identical. The 82–99 peptide within MBP has demonstrated high immunogenicity, perhaps because it demonstrates high homology with numerous viral peptides (59). Indeed, this region of the MBP gene has been targeted in numerous therapeutic strategies for multiple sclerosis (7,29,69). Unfortunately, this differential inflammatory response prevents shiverer cells from serving as a simple control for myelination even when the status of the MBP gene is manipulated as the sole variable (72). It is most likely that the diminished immune response inherent to shiverer NPCs would serve to augment recovery, however (48); this suggests that myelination is likely the variable of greatest significance.

Mature Oligodendrocyte Preservation Did Not Enhance Function

We had hypothesized that NPC transplantation would increase the total number of oligodendrocytes. However, NPC transplantation did not increase overall mature oligodendrocyte numbers. It is also interesting that preservation of large numbers of oligodendrocytes with trophins and pharmacotherapy did not enhance functional recovery. In the absence of axonal sparing, however, it is likely that many preserved mature oligodendrocytes were excess and not contributing to myelination of an axon. These results are thus consistent with the fact that mature oligodendrocytes contribute little to remyelination as they are unable to dedifferentiate or migrate (17). Indeed, transplantation of mature oligodendrocytes is not associated with remyelination (63). These findings provide further support for the finding that oligodendrocyte precursor cells have a much more significant effect on functional recovery than those which are mature.

The Functional Significance of Remyelination by Endogenous Cells

There has been controversy as to the extent and importance of remyelination by endogenous cells and whether remyelination is an important therapeutic target for exogenous cells (20,35,39). As transplantation of shiverer NPCs led to poorer outcome than even an untreated SCI and as shiverer cells ensheathed host axons, there is a suggestion that shiverer NPCs may block remyelination by endogenous cells. If this is true, then remyelination by endogenous cells makes an important contribution to normal functional recovery following SCI. It may be possible to augment this process to enhance functional recovery (26).

Strategies to Enhance Functional Recovery

Our study demonstrated both beneficial and detrimental effects of our NPC transplantation protocol, and both may be targeted to enhance recovery. As myelination appears to be of greatest functional significance, efforts to further enhance remyelination are likely to bear fruit. Molecular signals that control myelination are increasingly understood (26,49) and may be exploited for functional benefit.

Conclusions

In our protocol, remyelination appears to be an important mechanism responsible for enhanced functional recovery. It is not clear if our results are paradigm specific; a detailed characterization of other models is needed, in particular those involving nonneural cell types. The suggested functional importance of remyelination is a key finding of our work and suggests that strategies to augment remyelination may lead to important functional improvements for patients with spinal cord injury.

Footnotes

Acknowledgments

The authors wish to thank the Craig H. Neilsen Foundation, the Cervical Spine Research Society, and the Canadian Institutes of Health Research, who provided funding for these experiments. G. Hawryluk was supported by fellowships from Johnson and Johnson Medical Products, the McLaughlin Centre for Molecular Medicine, and the Congress of Neurological Surgeons/American Association of Neurological Surgeons. The authors also thank B. Azad for assistance with animal care. The authors contributed to the designing (G.W.J.H., M.G.F.) and performing of the experiments (G.W.J.H., S.S., D.C., S.W., M.E., N.F.), the data analysis (G.W.J.H., S.S., D.C., S.W., M.C., M.G.F.), and the writing of the manuscript (G.W.J.H., M.G.F.) as described. The authors declare no conflicts of interest.

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An Examination of the Mechanisms by which Neural Precursors Augment Recovery following Spinal Cord Injury: A Key Role for Remyelination

Abstract

Keywords

Introduction

Materials and Methods

General Animal Care

Neural Precursor Cell Isolation and Culture

Experimental Protocol and Groups

Functional Testing

Histology and Immunohistochemistry

Western Blotting

Fluoro-Gold ™ Retrograde Axonal Labeling

Cell Counting

Statistical Analysis

Results

Neural Precursor Cell Survival Decreases with Time and Is Independent of Trophin Infusion or Pharmacotherapy

Trophin Infusion and Pharmacotherapy Are Associated with Gray Matter, White Matter, and Oligodendrocyte Sparing

Trophin Infusion Increases Cyst, Lesional Tissue, and Inflammatory Infiltrate

Functional Recovery Is Related to the Myelinating Ability of Transplanted Cells

Discussion

Neural Precursor Cell Survival Decreases with Time and Is Independent of Trophin Infusion or Pharmacotherapy

Trophin Infusion via an Osmotic Minipump Affects NPC Location

Trophins Do Not Contribute to Functional Recovery

Neuroprotective Effects Were Not Associated with Functional Recovery

Myelin Basic Protein Deletion Alters the Inflammatory Response to Neural Precursor Cells

Mature Oligodendrocyte Preservation Did Not Enhance Function

The Functional Significance of Remyelination by Endogenous Cells

Strategies to Enhance Functional Recovery

Conclusions

Footnotes

Acknowledgments

References