SDF-1 overexpression by mesenchymal stem cells enhances GAP-43-positive axonal growth following spinal cord injury

2.1Engineering of MSCs that overexpress SDF-1α

Cloning of the human SDF-1α was originally performed by Orimo and colleague (2005), and this clone was purchased from Addgene (Cambridge, MA) as a stab culture containing the cDNA copy on a pBABE-puro retroviral backbone. This pBABE-puro SDF-1α was a gift from Bob Weinberg (Addgene plasmid # 12270; (Orimo et al., 2005). The SDF-1α open reading frame was re-cloned from the obtained cDNA with the addition of Bgl-II restriction site and a synthetic translation initiation Kozak sequence (GCCGCCACC) on the 5’ N-terminus immediately before the ATG start codon (Kozak, 1987) and the BAM-HI restriction site at the 3’ C-terminus, with removal of the TAA stop codon. Similarly, a T2A peptide sequence, linked by restriction site Xho1 to the reporter gene, green florescent protein (GFP), was cloned with no restriction site on the 3’ end, and with the addition of BAM-HI to the 5’ end. Forward cloning primers to SDF-1α contained the sequence 5’-AGATCTGCCGCCACCATGAACGCCAAGGTCGTG-3’, while reverse SDF-1α cloning primers were designed as 5’-GGATCCCTTGTTTAAAGCTTTCTCCAGG. The T2A-GFP sequence was previously constructed in our lab and was re-cloned with forward primers 5’-GGATCCGGCAGTGGAGAGGGCAGAGGAAG-3’ and reverse primers 5’-TTACTTGTACAGCTCGTCCATGGCCGAGAG-3’. The polymerase-chain reaction (PCR) cloned gene fragments were ligated onto commercially available T-Easy cloning vectors using T4 DNA ligase (New England Bio-Labs, Ipswich, MA), transformed and grown in JM 109 strain E. coli, purified using a Qiagen Mini Prep spin kit (Qiagen Inc., Limberg, Neatherlands), and recombined through restriction digestions and subsequent ligations, to formulate the sequence of interest SDF-1α-T2A-GFP onto the T-easy vector within a single open reading frame. Following sequencing, PCR and restriction digestion conformation, the sequence of interest was again recombined into the MSCV-puromycin-resistant retroviral vector, followed by transformation and purification as described above. The completed viral vector was transfected using calcium chloride into the PT67 packaging cell lines (CloneTech Laboratories Inc., Mountain View, CA) for the establishment of a continual production of a replication-incompetent virus. Viral containing supernatants were used to establish MSCs that over-express either SDF-1α/GFP or just GFP alone. Once GFP expression was detected, an antibiotic selection was performed to obtain a pure population of transduced MSCs. Confirmation and quantification of SDF-1α overexpression was performed using real-time PCR (RT-PCR) by comparatively analyzing SDF-1α expression against MSCs alone, with mRNA templates obtained from homogenized MSC cell pellets.

2.2MSC and NSC culture

MSCs were obtained from the femur bone marrow of a 3-month-old adult male Sprague Dawley rat, and expanded in Modified Eagles Medium (MEM) alpha, containing 10% fetal bovine serum (FBS), 10% horse serum (HS) and 1% penicillin and streptomycin (P/S) (Invitrogen Co., Carlsbad, CA). MSCs were passaged at approximately 70–80% confluence, using 0.05% trypsin/ethylenediaminetetraacetic acid (EDTA) and subsequent deactivation using 20% horse serum (Invitrogen Co., Carlsbad, CA). Neural stem cells (NSCs) were isolated from whole brain extraction of rat pups at embryonic day-18 and expanded in media, consisting of 20-ng/μL epidermal growth factor (EGF), 20-ng/μL basic fibroblast growth factor (bFGF), 1X B27 supplement without vitamin A, 1X N2 supplement, 1X Glutamax, in Neurobasal-A. Neurospheres reached an average size of 200-300-μm in diameter and were passaged using Accutase (Sigma Aldrich CO., St, Louis, MO) to bring cells into a single cell suspension.

2.3Transwell migration assay

As SDF-1α is known to induce migration of NSCs both in vitro and in vivo, the chemotactic effects of SDF-1α secretion by the modified MSCs were examined using a transwell migration chamber. SDF-1-MSCs or unmodified MSCs were seeded at 100,000-cells per well in a 24-transwell system (Corning Co., Corning, NY) in Neurobasal-A medium with 1% P/S, and allowed to incubate for 12-hours post-seeding. At 12-hours post-seeding of the MSCs, an 8-μm pore diameter, poly-L-lysine-coated transwell insert was used to separate 200,000 Hoechst-labeled NSCs from the lower chamber. NSCs were passaged into single cell suspension using cell dissociation buffer (Sigma Aldrich CO., St, Louis, MO) and manual pipetting to avoid digestion of sensitive receptors. Prior to seeding NSCs, Hoechst 33258 was added to the culture at a concentration of 1/1000-μL for 30 minutes and washed 4 times by centrifugation and re-suspension in 50-mL PBS. Following NSC passage, NSCs were suspended in Neurobasal-A and 1% P/S at 2,000-cells/μL, and 100-μL of this cell suspension was placed within each transwell insert of SDF-1-MSCs, unmodified MSCs, and no-cell control wells (n = 4). At 12-hours post-NSC-seeding, images were taken of the lower wells and all suspended and adhered cells were collected by enzymatic dissociation, using 0.05% trypsin EDTA. Cells were pelleted and suspended in 500-μL PBS and fluorescently Hoechst-labeled nuclei were counted with a hemocytometer and presented as cells/μL.

2.4Spinal cord injury

Contusion injuries, using a 25-mm drop from the NYU MASCIS Model II Impactor, were performed on 3-4 month old female Sprague Dawley rats, which weighed between 250 and 350 grams. Under sterile surgical conditions, rats were anesthetized and maintained under isofluorane for the duration of the procedure. Backs of the rats were shaved and sterilized with chlorohexadine, before making a 2.5–5 cm incision over the T8 vertebrae. Fat, fascia, and muscle were retracted from the dorsal process of the T8 vertebra before performing a laminectomy, ensuring that the dura remained intact. Additional muscle was retracted from the T7 and T9 vertebrae to allow for stabilization of the rat via vertebral clips before dropping the 10-gram weight onto the dorsal surface of the spinal cord. A small globule of subcutaneous fat was dissected and placed over the surface of the dura to help prevent fibrosis for re-exposure, and the muscles, fascia, and skin were sutured. Rats were given bimodal analgesia consisting of 0.02 mg/kg buprenorphine (Reckitt Benkiser Pharmaceuticals, Richmond, VA), and 1.0 mg/kg meloxicam (Boehringer Ingelheim Vetmedica Inc. St. Joseph, MO) twice a day, for three days following injury. Animals were housed in pairs, except for the first three days post-injury, at which time they were housed in individual cages, and were monitored for post-operative complications. Issues arising post-operation were limited to rare occasions of urinary tract infections, which required the administration of a three-day treatment of 10 mg/kg Enrofloxacin (Baytril; Med-Vet International, Mettawa, IL). Manual bladder expressions were performed four times per-day for the first week post-injury, and twice per day there-after.

2.5Transplantation into the spinal cord

Nine-days post-operation, animals were anesthetized via isofluorane, and the lesion site was re-exposed under sterile conditions. A 26-gauge Hamilton syringe, containing five microliters of either 300,000 cells (passaged between 35–43 times) and suspended in Hanks Buffered Saline Solution (HBSS), or HBSS alone, was lowered approximately 1.5 mm ventral to the dura at approximately a 45-degree angle, relative to the surface of the spinal cord and just left of the central vein, into the lesion epicenter. Five microliters were injected at a rate of 0.5μl/min, and the needle was maintained inside the spinal cord for at least three minutes post-injection. The rats were supported using vertebral clips to minimize respiratory movements during transplantation. Following transplantation, the muscles, fascia and skin were again sutured and the animals were given 1.0 mg/kg meloxicam for three days post-operation. Prior to transplantation, rats were assigned to three groups based on BBB scores obtained at one-week post-injury, to ensure that all groups had baseline scores within 0.25 standard deviations from each other.

2.6Behavioral analysis

One-week prior to injury, rats were acclimated to a circular plastic open-field (90 cm diameter) from which the BBB measurements were obtained. BBB scores were taken one-day prior to injury, and once each week following SCI, for seven-weeks, prior to euthanasia. As indicated above, each rat was assigned to one of the three study groups: SDF-1-MSC (n = 18), MSC (n = 17), or vehicle (n = 19), as well as a non-injured control group (n = 5). For BBB assessments, the rats were allowed to freely explore the open field for up to four minutes while two experienced raters evaluated the motor abilities. A small sub-sample of rats from each group was randomly selected to be allowed to survive for up to 15 weeks to determine the safety and long-term effects of the stem cell transplant. For this chronic-survival period, 4 rats were selected in the SDF-1-MSC-treatement group, while 5 rats were selected for the MSC-only group, and 3 rats were selected from the vehicle-treated group.

2.7Histology

At seven weeks post-injury, rats were euthanized via overdose of pentobarbital (Fatal Plus, Med-Vet International, Mettawa, IL) and transcardially perfused, using 4% formalin prepared from paraformaldehyde (Sigma Aldrich CO., St, Louis, MO). Following perfusion, the spinal cords were extracted and post-fixed overnight in 4% formalin, before being transferred to 30% sucrose (Sigma Aldrich CO., St, Louis, MO). Once the tissue was equilibrated to a 30% sucrose solution the spinal cords were cut into 2-cm longitudinal sections and frozen in methyl butane (Sigma Aldrich CO., St, Louis, MO), prior to short-term storage at – 80°C. Tissue was later sectioned, sagittal, at a 25-μm thickness and placed into 0.1 M phosphate-buffered in saline (PBS), and these free-floating sections were labeled using standard immunohistochemical procedures.

As the total white matter sparing around a cross-section of the lesion has been associated with the strongest correlate to behavior (Basso et al., 1995), tissue from a small random sample from each group (SDF-1-MSC, n = 4; MSC, n = 5; vehicle, n = 6) was cut in coronal cross-sections for white matter analysis. Sections of spinal cords were cut into 1-cm longitudinal segments, immersed in M-1 Embedding Matrix (Invitrogen Co., Carlsbad, CA), cut cross-section at 30-μm, and directly mounted as serial sections spaced 360-μm apart.

Astrogliosis and scarring were analyzed with antibodies against glial fibrilary acetic protein (Rb x GFAP; 1:1000; ab7260; AbCam Co., Cambridge, UK), and chondroitin sulfate proteoglycans (Ms x CSPG; 1:2000; MAB1581; Millipore Co., Billerica, MA). Inflammation was evaluated using antibodies against ionized calcium-binding adaptor molecule 1 (Rb x IBA-1; 1:9000; 019-19741; Wako Co., Richmond, VA), and white matter sparing was characterized by histological processing with eriochrome cyanine (Sigma Aldrich CO., St, Louis, MO). Immunohistochemistry was developed with avidin/biotin complex (ABC) with horseradish peroxidase (HRP) (Vector Laboratories Inc., Burlingame, CA) and diaminobenzidine (DAB) with nickel enhancement. The tissue was analyzed using optical densitometry using the NIH image analysis software, Image J. To evaluate the growth of axons inside the lesion site, immunohistochemistry labeling against growth-associated protein-43 (GAP-43; Rb x GAP-43; 1:500; EP890Y; AbCam Co., Cambridge, UK) was performed using ABC/DAB, with nickel enhancement. The region inside the lesion of whole tissue sections was imaged using a stereo-microscope (Nikon Optiphop-2 containing a JAI CV-S3200 camera; Nikon Co., Tokyo, Japan) and analyzed with Image J. Fluorescent immunohistochemistry labeling of GAP-43 was done to confirm that the localization of the label was inside the lesion site.

For analysis of tissue labeled with eriochrome cyanine, GFAP, CSPG, and IBA-1, densities of regions on the longitudinally sliced tissue sections (∼1 cm away from the lesion epicenter) were measured and used to normalize the densitometry results. Eriochrome cyanine staining on cross-sections were analyzed by tracing the spared white matter, and identifying the section containing the smallest spared volume for identification of the lesion epicenter. This lesion epicenter was used for directly comparing the total area of spared white matter at defined distances rostral and caudal to the lesion. For longitudinal sections, the image containing a 5255-μm-long section of tissue surrounding the lesion site was analyzed. All images acquired were processed and analyzed using blinded volunteers. Sagittal GFAP-labeled sections were analyzed for staining densities by tracing around the lesion, excluding cavitation and tissue inside the glial scar borders, while sagittal CSPG- and IBA-1-labeled sections were analyzed by tracing all tissue, excluding cavitation, and sagittal eriochrome cyanine sections were analyzed as whole tissue sections, including the lesion cavity. All data from sagittal GFAP-, CSPG-, IBA-1- and eriochrome cyanine-labeled tissue was presented as a percent optical density, standardized to regions at approximately 1-cm from the lesion epicenter. For sagittal GAP-43-labeled sections, images at the lesion epicenter were taken with a 10x objective, using standard light microscopy and the region inside the penumbra (tissue and cavitation) was traced and reported as mean optical density. For all optical density measurements, lower values suggest darker images, and more labeling/staining. To estimate total cavitation, the area of the lesion was determined by identifying the GFAP scar borders in sagittal sections, and the total area was traced, followed by tracing tissue inside the lesion and measuring the percent cavitation (see Fig. 7). Lesion boundaries were clearly visible in both GFAP and GAP-43 labeled sections which was characterized by the abrupt transition of tissue absent in stain (see Fig. 7).

Fig.1

PCR, Digestion, and Fluorescent Expression. (A) Genomic DNA from modified MSCs was isolated and examined with PCR using primers designed to flank the multiple cloning site, to analyze the insertion of the SDF-1-T2A-GFP sequence (Left), using the plasmid DNA (Middle) and GFP only plasmid (Right) as size controls. The genomic DNA revealed size-matched PCR products to the plasmid and a ∼300 larger base-pair fragment, compared to the GFP control. (B) Restriction digestion was performed on the completed MSCV-puro retroviral vector with the SDF-1-T2A-GFP sequence (Left), or just GFP sequence (Right) inserted into the multiple cloning site between Bgl-II and EcoRI restriction sites. (C) GFP was used as a reporter gene to confirm transcription and translation of the engineered sequence. GFP expression was confirmed using fluorescent microscopy with a FITC filter. Scale bar represents 50-μm.

Fig.2

SDF-1 RT-PCR Expression. The 2-^ΔΔCt method was applied to the results from RT-PCR of SDF-1-MSC cDNA and MSCs that express GFP-alone (GFP-MSC) cDNA, comparing the relative SDF-1 mRNA expression to unmodified MSCs. Results demonstrate a 400-fold increase in gene expression in SDF-1-MSCs compared to unmodified or MSCs that express GFP-alone. *Significance differences found at (p < 0.05) between SDF-1-MSC and MSC ΔΔCT values, relative to unmodified MSCs.

Fig.3

Transwell Migration Assay. Representative images acquired from the center of the wells following the transwell migration assay revealed significantly greater Hoechst-labeled nuclei in SDF-1-MSC wells (A), compared to MSC-only wells (p < 0.001; B) and non-MSC control wells (p < 0.001), while MSC-only wells contained significantly more Hoechst-labeled cells than in non-MSC control wells (p < 0.05). At 12 hours post-seeding with Hoechst pre-labeled NSCs, all cells in the bottom chamber were collected and re-suspended in 500-μL of PBS, and a 10-μL representative sample was collected and manually counted using a hemocytometer. Only Hoechst-labeled nuclei were counted, which revealed significant differences between all wells (p < 0.001; C). Image of non-MSC control wells is not shown due to the absence of cells in the lower chambers. Scale bars represent 200-μm. ^*p < 0.01, compared to non-MSC control wells. ^#p < 0.05, relative to MSC wells.

Fig.4

Behavioral Results. Prior to transplantation at 9-days post-injury, rats were distributed into groups to ensure an equal baseline mean (BBB scores ranged from 1–9). (A) When analyzing rats with the full range of baseline scores, transplantations of SDF-1-MSCs or MSCs-alone did not improve motor performance over the 7-weeks post-injury assessment period. (B) When rats with more severe injuries were removed from analysis (BBB scores ranged from 3–9), significant improvements were found with SDF-1-MSC treated rats. ^*p < 0.05 compared with vehicle-treated rats. ^†p < 0.005 compared with all other groups.

Fig.5

Immunohistochemistry GFAP, CSPG, Eriochrome Cyanine, IBA-1. (A-I) Immunohistology was performed to analyze major histopathological outcomes for astrocytes as measured by GFAP (A-C), for proteoglycans as measured by CSPG (D-F), for white matter pathology as measured by eriochrome cyanine staining (G-I), and for inflammation as measured by IBA-1 (J-L). No significant differences could be detected between SDF-1-MSC (A,D,G,J), MSC-only (B,E,H,K), or vehicle (C,F,I,L) groups. Areas of the lesions can be identified by clear boundaries in CSPG and GFAP labeling. Tissue within the lesion area is void of immunological labeling for GFAP, but dense in CSPG labeling. Scale bars represent 500-μm.

Fig.6

Cavitation. Immunohistochemical labeling against GFAP reveals distinct borders between healthy, and scared lesion tissue (A). Tissue existing within the compromised area was traced (B,D), followed by tracing the area of cavitation (C). A one-way ANOVA was used to compare total lesion area (D), as well as the percent cavitation (E). No differences were found in the total lesion area, while differences were found between groups for percent cavitation (p < 0.05), with Fisher’s PLSD post-hoc analysis revealing significant differences between SDF-1-MSC-treated rats and the vehicle-treated groups (p < 0.05). ^*p < 0.05 compared with vehicle-treated rats.

Fig.7

White Matter Sparing. One-way ANOVAs revealed differences in the area of spared white matter at 1080-μm from the lesion epicenter between MSC-only-, and vehicle-treated rats. No significant differences were found at any other location. Spinal cords were cut serially in cross sections, spaced 360-μm apart, and analyzed for white matter surrounding the lesion. Sections containing the least spared white matter were considered the epicenter, and distances up to 2,160-μm rostral and caudal to the epicenter were analyzed for each animal.

For analyzing tissue histopathologies in sagittal cut sections, every twelfth tissue section (cut serially and spaced 300-μm apart) was mounted and analyzed for each animal (SDF-1-MSC, n = 7; MSC-only, n = 7; Vehicle-treated, n = 7). Tissue sections disrupted during the sectioning, mounting or staining process were not included in analysis. For GFAP-labeling, an average of 4.73 (SD = 1.13) sections-per-animal were analyzed. For CSPG- labeled sections, an average of 4.95 (SD = 0.83) sections-per-animal were analyzed. For eriochrome-cyanine-stained sections, an average of 5.54 (SD = 0.97) sections-per-animal were analyzed. For IBA-1-labeled sections, an average of 4.93 (SD = 0.84) sections-per-animal were analyzed. For GAP-43-labeled sections, only tissue samples containing lesion boundaries were traced, averaging 4.01 (SD = 1.12) sections per-animal. For analyzing cavitation, only tissue samples containing distinct GFAP-stained boundaries were traced, averaging 4.10 (SD = 1.10) sections per-animal.

2.8Statistical analysis

For BBB measurements, the non-parametric Kruskall-Wallis analysis of variance (ANOVA) was utilized to compare data obtained from non-injured, SDF-1-MSC, MSC-only, and vehicle treatment groups for 7-weeks post-injury. For the sub-group of rats allowed to survive for 15-weeks post-injury, the Friedman’s non-parametric repeated measures ANOVA was used to determine whether changes occurred within each group from 7-weeks to 15-weeks post-injury. A one-way ANOVA was used for comparing SDF-1-MSC, MSC-only, and vehicle treatment groups on all other measures, using Statistical Package for the Social Sciences version 23 (SPSS). When appropriate, Fisher’s Protected Least Significant Difference (PLSD) test was used for post hoc analysis of all parametric data. To determine whether meaningful relationships may exist between outcome measures, one-tailed Pearson correlations were performed between BBB scores at 7-weeks post-injury, measures of cavitation, as well as densities in GAP-43, GFAP, CSPGs, and eriochrome cyanine staining.

3.1Cloning and construction of SDF-1α-T2A-GFP onto MSCV-puro

Cloning of SDF-1α, T2A-GFP, and recombination onto the MSCV vector were performed successfully, without mutation, as indicated by gene sequencing. Restriction digestion and PCR conformation was used to evaluate the size and orientation of the completed plasmid (Fig. 1 A-B). PT67 cells were successfully transfected before selection in puromycin to obtain a pure population of virus-producing cells.

3.2Transduction and expression of SDF-1α in MSCs

Viral supernatants of GFP- and SDF-1α-GFP producing PT67 cells were obtained after 24-hours in culture and used to infect MSCs. One day later, GFP expression in both cell lines could be detected under fluorescent microscopy (Fig. 1C). Again, the population of cells was purified with puromycin, and the culture was allowed to recover. DNA and mRNA were obtained for analyzing the insertion and expression of the transgene with PCR, which demonstrated successful integration into the cells (Fig. 1A) and RT-PCR demonstrating a significant (F(2,3) = 12.99, p < 0.05) 400 fold increase (Mean difference = 414.83, SD = 135.75) in SDF-1α expression compared to non-transfected MSCs using the 2^-ΔΔCt method (p < 0.05; n = 2) (Fig. 2). A 2^-ΔΔCt value of 8.856 between SDF-1α expressing MSCs (SDF-1α Ct = 20.65, GAP-DH Ct = 19.27) and non-transfected MSCs (SDF-1α Ct = 27.06, GAP-DH Ct = 17.05) was obtained, confirming transcription of the virus. A 2^-ΔΔCt value of – 0.14 between GFP transfected (SDF-1α Ct = 27.79, GAP-DH Ct = 17.05) and non-transfected MSCs demonstrated that transfection, alone, does not affect the expression of SDF-1α.

3.3Function of SDF-1α overexpression by MSCs in vitro

The preservation of function by this cloned SDF-1α was confirmed using the transwell chemotaxis assay. Results from this migration experiment demonstrated significant differences between groups, using a one-way ANOVA (F(2,9) = 52.36, p < 0.001), with more NSCs migrating into the lower chamber of SDF-1-MSC-wells (M = 246.25, SD = 29.18) compared to both the MSCs (M = 98.75, SD = 3.14; p < 0.001) and no-MSC control wells (M = 2.50, SD = 1.44; p < 0.001), while MSC-alone wells also contained significantly more NSCs than no-MSC control wells (p < 0.005; Fig. 3).

3.4Behavior

Behavioral analysis, using Kruskal-Wallis non-parametric analysis of variance, revealed significant between-group differences at all time points (p < 0.005), however, as determined following individual Mann-Whitney U tests, differences only existed between non-injured controls and other treatment groups (p < 0.005). At 7-weeks post-injury, a trend indicating improved function of rats receiving transplants of SDF-1-MSC was observed when compared to vehicle-treated rats (U = 108.50, p = 0.054). Further analysis, using a sub-group of rats with less severe injuries, and for which rats with BBB scores less than 3 at the time of transplantation were omitted from analysis, revealed a significant treatment effect at 7-weeks post-transplantation between the SDF-1-MSC- and vehicle-treated rats (U = 52.50, p < 0.037) (Fig. 4). Analyses of the rats that were allowed to survive for 15-weeks post-injury were analyzed bi-weekly for changes in BBB scores from 7-weeks to 15-weeks using the Friedman’s test, and showed no further improvements beyond 7-weeks post-injury.

3.5Histology

No between-group differences were found for optical densitometry measurements on longitudinal sections for the GFAP-labeled sections (F(2,17) = 0.268, p = 0.768; Fig. 5A-C), CSPG-labeled sections (F(2,17) = 0.493, p = 0.619; Fig. 5D-F), eriochrome-cyanine-stained sections (F(2,17) = 0.105, p = 0.901; Fig. 5G-I), or IBA-1-labeled sections (F(2,17) = 1.006, p = 0.386; Fig. 5J-L). For analyzing tissue histopathologies, every twelfth tissue section (cut serially and spaced 300-μm apart) from each animal was mounted and analyzed.

Regions immediately surrounding the lesion epicenter were filled with dense, CSPG-rich tissue, with hollow, fluid-filled, cavitation existing around the scar. To measure the extent of cavitation surrounding the lesion area, the total area of cavitation was determined by tracing around regions of compromised tissue in sagittal sliced, GFAP-labeled sections, followed by tracing cavitation on image J and computing the percent of the injured area that was filled with tissue (Fig. 6). Tissue samples containing distinct GFAP-stained boundaries were traced. A one-way ANOVA failed to detect differences in the total lesion area (F(2,16) = 0.433, p = 0.656). However, between-group differences on the percent of lesion area filled with cavitation (F(2,16) = 3.706, p < 0.05) were found (Fig. 6E). Fisher’s PLSD post-hoc analyses indicated that SDF-1-MSC-treated rats had less cavitation within the lesion area than didvehicle-treated rats, but no differences were found between MSC-only and vehicle-treated rats, or between SDF-1-MSC and MSC-only-treated rats (Fig. 6E).

A one-way ANOVA revealed between group differences in the total area of white matter sparing at 1080-μm caudal to the lesion epicenter (F(2,14) = 4.399, p < 0.05; Figs. 7 and 8), in cross-sectional analysis of eriochrome-cyanine labeled sections. Fisher’s PLSD post-hoc analysis indicated that MSC-only-treated rats (M = 1148.58, SD = 587.61) had greater white matter areas compared to vehicle-treated rats (M = 787.49, SD = 641.015; p < 0.05). No differences in white matter sparing were found at any of the other measured locations.

Fig.8

Eriochrome Cyanine Cross-Section. White matter sparing was analyzed using eriochrome cyanine staining, revealing graded myelin loss surrounding the lesion epicenter. Spinal cord cross sections were cut serially, spaced 360-μm apart in the rostral-caudal direction, and directly mounted. Sections were analyzed by tracing dense blue boundaries to evaluate spared white matter and compared at each representative distance from the lesion epicenter.

To characterize the effects of SDF-1-MSCs on the growth response of axons, GAP-43 was immunologically labeled with ABC/DAB tags, followed by imaging the lesion centers with a 10x objective (Fig. 9). Only tissue samples containing lesion boundaries were traced. Tissue existing within the lesion center was identified and traced using Image J software and the optical densities were analyzed using a one-way ANOVA, revealing significant between-group differences (F(2,17) = 5.428, p < 0.05). The Fisher’s PLSD post-hoc analysis indicated that SDF-1-MSC-treated rats had greater GAP-43 densities than did vehicle controls (M = 201.49, SD = 16.66; p < 0.005) (Fig. 10), but were not significantly different than MSC-only rats.

Fig.9

Immunohistochemistry GAP-43. GAP-43 immunological labeling was found to be significantly increased within the lesion cores for SDF-1-MSC-treated (A,D,G), but not in MSC-only rats (B,E,H) compared to vehicle-treated controls (C,F,I). Data was quantified using DAB labeling (G-I) by tracing lesion cores and obtaining optical densitometry measurements using Image J. Fluorescent microscopy was utilized for imaging to confirm the specificity and localization of labeling (A-F). A 10x objective (A-C, G-I) revealed GAP-43 labeling predominately within the lesion center, while images taken with 20x objectives (D-F) revealed individual fibers or fiber bundles. Scale bars represent 100-μm (A-C, G-I) and 50-μm (D-E).

Fig.10

GAP-43 Optical Density. GAP-43 immunological labeling was done using ABC/DAB, followed by tracing the lesion centers and evaluating optical densities. Brighter images reveal less labeling, demonstrating significantly greater GAP-43 densities in SDF-1-MSC-treated rats compared to vehicle controls (p < 0.005), but not between MSC-only and vehicle controls, or SDF-1-MSC and MSC-only rats. ^*p < 0.05, relative to vehicle-treated rats.

3.6Correlation of outcome measures

Significant negative correlations were found between end-point BBB scores and GAP-43-labeling densities (R = – 0.420, p < 0.05) for all rats in the first analysis, but not for rats with the less significant injuries (R = –0.287, p = 0.141). This analysis suggests that animals with higher BBB scores also contained more GAP-43 labeled fibers within the lesion. Significant negative correlations also existed between densities of eriochrome cyanine staining and GFAP- (R = –0.609; R = –0.615; p < 0.05), and CSPG-labeling (R = –0.469; R = –0.473; p < 0.05) for all rats and from the sub-group of rats were less deficits, respectively. This suggests that less myelin surrounding the lesion correlates to more labeling with GFAP and CSPGs. Significant positive correlations were found for rats from both analyses between GAP-43 labeling and cavitation (R = 0.554; R = 0.723; p < 0.01), which suggest that a greater amount of cavitation may result in less GAP-43 labeling within the lesion. Significant correlations were not found between functional outcomes and the amount of cavitation for analyses with either all rats or the sub-group with less severe deficits.