Improved Healing after the Co-Transplantation of HO-1 and BDNF Overexpressed Mesenchymal Stem Cells in the Subacute Spinal Cord Injury of Dogs

Selection of Animal

The experiment was performed on 16 male dogs between the ages of 1 and 2 years, with an average weight of 10.5 ± 1.56 kg. The dogs were randomly divided into four groups, comprising four dogs each. The groups were assigned according to the type of cells used. Combination group – both BDNF and HO-1 overexpressed Ad-MSCs were used in combination; BDNF-MSCs group – only BDNF overexpressed Ad-MSCs were used; HO-1-MSCs group – only HO-1 overexpressed Ad-MSCs were used; GFP-MSCs group – only Ad-MSCs expressing green fluorescent protein (GFP) were injected at the injury site and were used as a control. The dogs were kept in accordance with the guidelines of the Institute of Laboratory Animal Resources, Seoul National University, Korea. The research was approved by the Animal Care and Use Committee of Seoul National University (SNU-160720-13). All the dogs were clinically sound and showed no neurological abnormality.

Isolation and Culture of ADSCs

The gluteal subcutaneous fat was aseptically collected from healthy beagle dog of age 1.5 years, under general anesthesia. The tissues were washed with phosphate buffer saline (PBS), minced in a six-well plate, and digested with collagenase type-1 (1 mg/ml, Sigma-Aldrich, St. Louis, MO, USA), for 2 h under shaking incubation. The suspension was strained through a nylon mesh of pore size 100 µm and centrifuged to obtain the stromal vascular fraction (SVF). The SVF was resuspended and cultured in commercially available low-glucose Dulbecco’s modified Eagle’s medium (DMEM; GenDEPOT, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (FBS; Gibco BRL, Grand Island, NY, USA), and 1% penicillin and streptomycin (PS), for 24 h in a humidified incubator at 37°C and 5% CO₂. After 24 h, the Ad-MSCs were washed with PBS to remove the cells debris, and the medium was changed. Next time the medium was changed after an interval of 48 h, until the cells reached 80–90% confluence. The cells were either stored or subcultured, and further processed for the experiment ¹⁶ .

Preparation of Transgenic Lentivirus and Ad-MSCs Transduction

Ad-MSCs were transduced with lentivirus vectors encoding GFP, canine BDNF with GFP, or canine HO-1 with GFP, depending on the group to which the cells belonged. For the preparation of the edited plasmid with the desired gene, the canine HO-1 and BDNF genes were cloned by referring to the Pub-Med database. The genes encoding Flag-tagged BDNF, HO-1, and GFP were amplified by Phusion DNA Polymerases with the cDNA of canine peripheral blood (Thermo Scientific, Pittsburgh, PA, USA). The canine-specific primer set for BDNF (forward, 5′-ATGACCATCCTTTTCCTTAC-3′; reverse, 3′-GATAGAAGGGGAGAATTACC-5′), and HO-1 (forward, 5′-GACAGCATGCCCCAGGAT-3′; reverse, 3′-TCACAGCCTAAGGAGCCAGT-5′), with restriction enzymes EcoRI and BamHI, were inserted into a pCDH-EF1-MCS-pA-PGK-copGFP-T2A-Puro vector (System Biosciences, Mountain View, CA, USA). Dharmacon Trans-Lentivirus packaging system was used to prepare the transgenic viruses (GE Healthcare, Lafayette, CO, USA). First, the HEK293 T cells were cultured in a 100 mm culture plate using a high-glucose medium (DMEM) containing 10% non-heat-inactivated FBS and 1% PS, until the cell density of 5.5 × 10⁶ cells in 14 ml was reached. The suspension of DNA transfer vectors encoding GFP, BDNF, and HMOX-1, the lentiviral packaging mix (Fisher Scientific Cat #14-432-23) encoding viral proteins Gag-Pol, Rev, and VSV-G, and CaCl₂, was made and added, drop-wise, to the cultured cells for transfection. The cells were incubated for 10–16 h at 37°C and 5% CO₂. The medium was changed and the viral particles were collected from the culture medium after 48 h of incubation. Successful transfection was confirmed by observing GFP expression during incubation under a fluorescent microscope. Finally, three types of lentiviruses, Lenti-BDNF-GFP, Lenti-HO-1-GFP, and Lenti-GFP, were obtained for gene delivery into the Ad-MSCs. The concentration of viral particles was determined according to the manufacturer’s guidelines. The MSCs were transduced at passage 1, during 50–60% confluence, by the lentivirus at MOI = 100 ¹⁷ , and were subcultured. The transduced cells were cultured with 3 μg/ml puromycine to get a higher percentage of transduced stem cells. We obtained three types of cells: Ad-MSCs overexpressing BDNF (BDNF-MSCs), Ad-MSCs overexpressing HO-1 (HO-1-MSCs), and Ad-MSCs expressing only GFP (GFP-MSCs). The modified stem cells were subcultured up to three passages and were subsequently used for in vivo study.

Induction of SCI

The SCI was induced by the balloon compression method under general anesthesia ¹⁸ . A mini hemi-laminectomy was performed by creating 3–5 mm hole at the fourth lumbar vertebral arch (L4) using a high-speed pneumatic burr. A 6 Fr embolectomy catheter (Sorin, Biomedica, Salugia, VC, Italy) was inserted and advanced to the cranial margin of the first lumbar vertebra (L1), using fluoroscopic guidance. The balloon was distended with contrast agent (Omnipaque, Amersham Health, Carrington Hill, Ireland) diluted in normal saline (50:50) at a dose rate of 50 µl/kg body weight. The catheter was removed after 12 h. This technique was found to be reliable and repeated with satisfactory outcomes in our previous studies ^{12,14,18 –20} . As found by computed tomography, this SCI model occludes 80% of the spinal canal which inflicts compression injury to the spinal cord ^12,18 . All the dogs were kept under close supervision during the experimental period. Each dog was placed in a big cage with soft bedding comprising 12 mm thick soft mats to prevent bed sores. Food and water were provided ad libitum. Manual bladder compression was performed three times a day to assist with urination.

Transplantation of Overexpressed Ad-MSCs

After 1 week of SCI, BDNF-MSCs, HO-1-MSCs, and GFP-MSCs were injected. The injured segment of the spinal cord at L1 was exposed through dorsal laminectomy, and about 1 × 10⁷ cells diluted in 150 µl PBS were injected 2–3 mm deep into the spinal cord parenchyma. Three injections of 50 µl each were made: two at the epicenter and one in the center of the injured spinal cord segment. The cells were injected slowly, within an interval of 5 min. In the combination group, we mixed 5 × 10⁶ BDNF-MSCs with 5 × 10⁶ HO-1-MSCs to make a full dose of 1 ×10⁷ cells. After Ad-MSCs transplantation, the dogs were kept for 8 weeks and observed for functional improvement.

Behavioral Assessment

All the dogs were evaluated for gait analysis before SCI and after Ad-MSCs injection. The motor activity was assessed by 19-point scoring system of canine Basso, Beattie, and Bresnahan (cBBB) ²¹ . All the dogs were observed for improvement in functional recovery on a daily basis, when they were resting in the cage or set free during cleaning and washing of the cage. On a weekly basis, the improvement in the cBBB score was recorded. Each dog was allowed to move freely in a contained area for 4-5 min to observe improvement in locomotion, weight-bearing ability, and hindlimb coordination. The movement of the joints and muscle contraction was recorded when the dog’s hindquarters were lifted with the base of the tail while standing, and flexion of the joints was recorded while the dog was lifted by an attendant. For deep nociception, the phalanges were pinched with an artery forceps and the degree of pain perception was evaluated by the degree of limb protraction, lip licking, vocalization, and facial expressions. The gait evaluation was performed by three persons blind to the study and the average values were presented for each dog and group.

Western Blot Analysis

At the end of the experimental period, each dog was euthanized and the spinal cords were extracted through dorsal laminectomy, from the twelfth thoracic vertebra (T12) to the third lumbar vertebra (L3). The spinal cord segments were immersed for 12 h in 10% sucrose at 4°C, followed by 20% sucrose at 4°C for the next 24 h. The duramater was carefully removed and the spinal cord segments were embedded and frozen in the optimal cutting temperature compound (Rica Biosystems, Richmond, VA, USA). The spinal cord segments were longitudinally incised into two equal halves; one half was kept for western blot analysis and the other half was employed for cryosectioning, for performing immunofluorescent staining, and histopathological assessment.

For western blotting, the entire injured segment of the spinal cord tissue was properly minced and incubated with 300 μl Ripa Lysis Buffer (Gen Depot,Grand Island, NY, USA), and 3 μl proteinase inhibitor solution (Gen depot) for 30 min on ice, followed by cold centrifugation (4°C, 10 min, 13,000 rpm). In the case of transgenic cells, for the confirmation of successful expression of the transfected gene, the cells of each treatment group were harvested at 90% confluence from three 150 mm petri dishes, pelleted, incubated with Ripa Lysis Buffer, and centrifuged (4°C, 10 min, 13,000 rpm). The supernatant was collected and the protein concentration was determined by the Bradford assay. About 20 μg of protein of each sample was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electrically transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% skimmed milk for 1 h, followed by overnight incubation with primary antibodies. The following primary antibodies were used: for neural markers, Tuj-1 (sc69966, β-III-tubulin, immature neurons), neurofilament (NF-M, mature neurons, sc-398532), GAP-43 (growth associated proteins for neuro-regeneration, ab-12274), GFAP (astrocytes, sc65343), nestin (neural differentiation, neural stem cells, ab7695); for inflammatory markers, COX2 (cyclooxygenase-2, sc19999), TNF-α (sc1350), IL-6 (interleukin-6, ab-6672), phosphorylated STAT3 (sc1008 R); for anti-inflammatory markers, IL-10 (interleukin-10, R&D, MAB7352), HO-1 (ab-13243); and for neurotrophic factor, BDNF (ab-101747). The membrane was incubated for 1 h at 4°C with anti-mouse secondary antibody (ab6728), and anti-rabbit secondary antibody (ab6721). The β-actin antibody (sc-47778) was used as an internal reference. Protein bands were visualized using Cyanagen WESTAR chemiluminescent substrates (Cyanagen, Bologna BO, Italy), through the image quant LAS 4000 mini system (GE Health Care Biosciences, Uppsala, Sweden).

Immunohistological and Histopathological Assessments

For immunofluorescent staining, the spinal cord segment was sliced at 10 μm by the cryomicrotome, at –20°C. The sections were mounted on silane-coated slides, fixed, and permeated for 10 min with 0.1% v/v Triton X-100, followed by an incubation with 1% bovine serum albumin in PBS for 30 min (BSA; Sigma-Aldrich). The slides were incubated with primary antibodies of neural marker Tuj-1, NF-M, GFAP, and nestin for 24 h at 4°C. This was followed by 2 h incubation of fluorescein iso-thio-cyanate conjugated anti-mouse (Alexa flour, ab-150111), and anti-rabbit secondary antibodies (Flamma 648). DAPI was used to stain the nuclei, and a coverslip was placed over the sections. The slides were examined under the microscope (EVOS FL Imaging System, Stanwood, Washington USA). Cells positive for specific markers were counted from the five randomly selected areas at the injury site, on a slide, through the integrated cell counting system in the microscope. The values were expressed as a percentage representing the average of each slide, and the relative group.

In order to measure the degree of fibrosis, hemorrhages, and vacuoles, slides were stained with H&E. The images were obtained to include the entire injured area of the spinal cord – that is, rostral, caudal, and middle segments of the injured spinal cords ²² . The lesions were measured for degree of fibrosis by computer-associated image analysis system (Image-J version 1.37; National Institute of Health, Bethesda, MS, USA). Average values were presented for each slide and group.

Statistical Analysis

The data was presented as a mean ± standard error (SE). All experiments were analyzed for the presence of general significance among the groups by a nonparametric two-way analysis of variance (ANOVA), and a post hoc Tukey’s HSD test was used to analyze the significant difference between two groups. A Student t test was used to examine the significantly increased expression of HO-1 and BDNF by HO-1-MSCs and BDNF-MSCs. OriginPro 8.5 software (Northampton, MA, USA) was used to analyze the data and P ≤ 0.05 was taken as a significant value. P value was expressed together with an F or Q value.

Behavioral Assessment

The functional outcomes of the use of modified Ad-MSCs was assessed using the cBBB score. All the dogs were physically normal with a cBBB score of 19 before the SCI. After SCI, the score for all the dogs was zero. The hindquarters of all the dogs were completely paralyzed, showing no deep pain. After 1 week of SCI, Ad-MSCs were injected (intrathecal), and at the time most of the dogs exhibited few reflexes. The cBBB score recorded at the time of Ad-MSCs transplantation was 0.25 ± 0.5 for combination, BDNF, and GFP-MSCs groups, and 0 for the HO-1-MSCs group. During the course of the experimental period the combination, BDNF, and HO-1-MSCs groups showed improvement in their BBB score up to an intermediate stage of improvement – that is, 8–13 – however, the GFP-MSCs group remained in the early stage of BBB score improvement at 0–7.

After 5 weeks the cBBB score become significantly different among the groups (two-way ANOVA F = 2.34, P ≤ 0.05) and remained significant after 6 weeks (two-way ANOVA F = 5.04, P ≤ 0.05), 7 weeks (two- way ANOVA F = 13.07, P ≤ 0.05), and 8 weeks (two-way ANOVA F = 2.34, P ≤ 0.05) (Figure 3). A Tukey’s post hoc test showed that the cBBB score was significantly improved by the combination group after 5 weeks (q = 4.43, *P ≤ 0.05), 6 weeks (q = 5.34, *P ≤ 0.05), 7 weeks (q = 8.83, *P ≤ 0.05), and 8 weeks (q = 7.72, *P ≤ 0.05) compared to the GFP-MSCs group. Compared to the HO-1-MSCs group, the combination group attained significant improvement in cBBB score after 7 weeks (q = 4.75, ⁺P ≤ 0.05) and 8 weeks (q = 5.40, ⁺P ≤ 0.05); compared to the BDNF-MSCs group it attained a significant level after 8 weeks of transplantation (q = 4.7, ^%P ≤ 0.05) (Figure 3). Comparing the BDNF group with the HO-1 group, both showed similar patterns of improvement in the cBBB score (q = 0.67, ns) after 8 weeks (Figure 3); comparing the BDNF-MSCs group with the GFP-MSCs group, a significantly higher BBB score was found after 7 (q = 4.75, ^$P ≤ 0.05) and 8 weeks (q = 5.33, ^$P ≤ 0.05). A significant improvement in the score of the HO-1-MSCs group was seen after 8 weeks (q = 4.66, #P ≤ 0.05) compared to the GFP-MSCs group (Figure 3). In the combination group, three out of four dogs were able to stand and support their body weight; they were able to support their own body weight when forced to walk as well, but coordination between the legs was poor. In the BDNF and HO-1 groups, only one out of four dogs was able to bear its weight. In the GFP-MSCs group, the dogs were only able to bear their weight when support was provided at the tuber coxae. Collectively, the average BBB score obtained after 8 weeks was 10 ± 0.8, 8.5 ±1, 8.25 ± 1.2, and 6.75 ± 0.5 for combination, BDNF, HO-1, and GFP-MSCs groups respectively. Dogs that were able to support their body weight showed stronger limb protraction upon phalangeal pinching; however, no dog vocalized during the pinching process, which may indicate spinal reflex action with weak recovery of pain perception. After SCI, all the dogs failed to urinate voluntarily. At the end of the experimental period, voluntary urination recovered in two dogs in the combination group, one dog each in the BDNF and HO-1 groups, and none in the GFP-MSCs group after 8 weeks of Ad-MSCs injection. Defecation was normal in all dogs.

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Figure 3.

Changes in BBB score until 8 weeks after transplantation of MSCs. The BBB score of the combination group, BDNF-MSCs group, and the HO-1-MSCs group was significantly higher. ^*P ≤ 0.05, combination vs. GFP-MSCs; ^%P ≤ 0.05, combination vs. BDNF-MSCs; ⁺P ≤ 0.05, combination vs. HO-1-MSCs; ^$P ≤ 0.05, BDNF-MSCs vs. GFP-MSCs; ^#P ≤ 0.05, HO-1-MSCs vs. GFP-MSCs. Each group represents the average score of four dogs (n = 4).

Neuroregeneration and Differentiation

The expression of Tuj-1, GAP-43, and NF-M was the highest in the combination group. A Tukey’s post hoc analysis showed a significant upregulated expression of Tuj-1 (q = 5.14, *P ≤ 0.05), GAP43 (q = 4.40, *P ≤ 0.05), and NF-M (q = 4.95, *P ≤ 0.05) in the combination group compared to other groups (Figure 4). It was the same in the BDNF and HO-1-MSCs groups; however, compared to GFP-MSCs, the BDNF and HO-1-MSCs groups showed significantly higher expression for Tuj-1 (q = 4.23, ^#P ≤ 0.05), GAP-43 (q = 5.39, ^#P ≤ 0.05), and NF-M (q = 6.52, ^#P ≤ 0.05). The expression of nestin, GFAP, and GALC was the same in the combination group and the BDNF-MSCs group; however, both groups showed a significantly higher expression of nestin (q = 6.71, *P ≤ 0.05), GFAP (q = 5.64, *P ≤ 0.05), and GALC (q = 5.13, *P ≤ 0.05) compared to the HO-1-MSCs and GFP-MSCs groups (Figure 4). The expression of GALC was also higher in the HO-1-MSCs group than in the GFP-MSCs group (q = 4.38, #P ≤ 0.05). Collectively, the process of neuroregeneration might be upregulated in the combination group, evidenced by the highest expression of Tuj-1, GAP-43, and NF-M, since an equal level of upregulated expression of nestin, GALC, and GFAP, was found in the combination and BDNF groups (Figure 4).

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Figure 4.

Expression of neural markers. (a) The densities of neural markers. (b) Quantitative data obtained by densitometry showing optical densities (ODs) relative to GFP-MSCs. The expression of Tuj-1, GAP-43, and NF-M was significantly increased in the combination group compared to the other groups (*P ≤ 0.05), while it was the same between BDNF and HO-1-MSCs groups, but significantly higher than the GFP-MSCs group (^#P ≤ 0.05). The expression of nestin, GFAP, and GALC was the same in the combination and BDNF groups, but significantly higher than the HO-1- and GFP-MSCs groups (*P ≤ 0.05). GALC was also significantly higher in the HO-1 group compared to the GFP-MSCs group (^#P ≤ 0.05). The graph represents mean ± standard error of four dogs per groups, as determined by densitometry relative to β-actin.

Expression of Inflammatory and Anti-Inflammatory Markers

The expressions of inflammatory markers were significantly reduced in the combination group. A Tukey’s post hoc analysis showed that the expression of TNF-α and IL-6 was significantly reduced in both combination and HO-1-MSCs groups compared to BDNF-MSCs (q = 4.12, *P ≤ 0.05) and GFP-MSCs (q = 4.27, *P ≤ 0.05) (Figure 5). Additionally, the expression was not different between the BDNF-MSCs group and the GFP-MSCs group (q = 1.13, ns). COX-2 represented similar expression within the combination, BDNF-MSCs, and HO-1-MSCs groups; however, this was lower than the expression in the GFP-MSCs group (q = 6.88, *P ≤ 0.05).

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Figure 5.

Expression of inflammatory and anti-inflammatory markers. (a) The densities of inflammatory and anti-inflammatory markers. (b) Quantitative data obtained by densitometry showing optical densities (OD) relative to GFP-MSCs. The expression of TNF-α and IL-6 was significantly lower in combination and HO-1-MSCs groups compared to BDNF and GFP-MSCs groups (*P ≤ 0.05). The expression of COX2 was significantly decreased in all combination, BDNF-, and HO-1-MSCs groups compared to the GFP-MSCs group (*P ≤ 0.05). pSTAT3 was upregulated in both combination and HO-1-MSCs group compared to BDNF- and GFP-MSCs groups (*P ≤ 0.05); its expression was also higher in the BDNF group compared to the GFP-MSCs group (^#P ≤ 0.05). IL-10 was significantly higher in the combination and HO-1 groups (*P ≤ 0.05) compared to the BDNF and GFP-MSCs groups. The graph represents mean ± standard error of four dogs per groups, as determined by densitometry relative to β-actin.

pSTAT3 was upregulated in the combination and HO-1 groups compared to the BDNF-MSCs and GFP-MSCs groups (two-way ANOVA F = 10.35, *P ≤ 0.05) (Figure 5). The combination group showed significantly higher expression of pSTAT3 compared to BDNF-MSCs (q = 7.56, *P ≤ 0.05) and GFP-MSCs (q = 7.46, *P ≤ 0.05). Within the BDNF-MSCs and GFP-MSCs groups, a higher expression in the BDNF-MSCs group was evidenced than in the GFP-MSCs group (q = 4.29, #P ≤ 0.05) (Figure 5). IL-10, as an anti- inflammatory marker, showed upregulation in both the combination and HO-1 groups, but remained downregulated in the BDNF and GFP-MSCs groups (two-way ANOVA, F = 9.56, *P ≤ 0.05) (Figure 5). A significantly higher expression of IL-10 was observed in the combination group compared to the BDNF-MSCs group (q = 6.26, *P ≤ 0.05) and GFP-MSCs group (q = 7.45, *P ≤ 0.05) (Figure 5).

Immunohistochemistry

Immunohistochemistry was performed to evaluate the expression of neural markers Tuj-1, GFAP, NF-M, and nestin in transplanted MSCs and host cells at the injured segment of the spinal cord. The transplanted cells survived until 8 weeks. They were evenly distributed in the injured spinal cord segment. Based upon the presence of GFP expression, the percentage of live transplanted cells observed after 8 weeks was 14.9 ± 5.8%, 11 ± 4.36%, 14.1 ± 4.65%, 10.89 ± 5.28% for combination, BDNF-MSCs, HO-1-MSCs, and GFP-MSCs groups respectively. No clustering of the transplanted cells was observed. The cells were found together with endogenous neurons and glial cells, because GFP-positive cells shown by green and blue color were found among the cells that were negative for GFP and positive for blue and red color of the neural marker.

Regarding the expression of neural markers, we found a similar pattern of expression, compared to that found in western blot results. We obtained the highest percentage of Tuj-1 positive cells, at 16.78 ± 2.14%, in the combination group compared to BDNF-MSCs (q = 5.93, *P ≤ 0.05), HO-1-MSCs (q = 7.65, *P ≤ 0.05) and GFP-MSCs (q = 8.64, *P ≤ 0.05). The percentage value was not significantly different between the BDNF- and HO-1-MSCs groups, at 10.17 ± 3.378% and 7.13 ± 2.86%, respectively, although it was higher than that of the GFP-MSCs group, at 4.34 ± 0.30% (q = 5.2 #P ≤ 0.05). The percentage expression of GFAP was similarly high in the combination group, at 16.87 ± 3.30% compared to the BDNF group, at 14.25 ± 1.21% (Figure 6). The percentage expression of GFAP in the combination group was higher than that of the HO-1 (q = 5.49, *P ≤ 0.05) and GFP-MSCs groups (q = 4.22, *P ≤ 0.05), at 4.9 ± 2.1% and 8.40 ± 5.40%, respectively. The BDNF-MSCs group showed a similar pattern of expression for GFAP compared to HO-1-MSCs and GFP-MSCs (Figure 6(a,b)). In case of NF-M-positive cells, the percentage was the highest in the combination group, at 13.50 ± 5.50% compared to BDNF-MSCs (q = 7.07, *P ≤ 0.05), HO-1-MSCs (q = 7.23, *P ≤ 0.05), and GFP-MSCs (q = 7.56, *P ≤ 0.05) (Figure 6(a,b)), followed by the BDNF- and HO-1-MSCs groups, at 9.70 ± 1.03% and 7.91 ± 0.30%, respectively compared to the GFP-MSCs group, at 6.27 ± 1.68% (Figure 6(a,b)).

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Figure 6.

Immunofluorescent staining for the expression of neural markers. (a) images of stained slides. The slides were stained with Tuj-1, NF-M, GFAP, and nestin as red, and the nucleus was stained with DAPI as blue. Each image represents the four samples per group with a scale bar of 200 μm. (b) Percentage expression of neural marker positive cells. The percentage of cells positive for Tuj-1 and NF-M was highest in the combination group, followed by higher percentages in BDNF- and HO-1-MSCs groups compared to the GFP-MSCs group (*^#P ≤ 0.05). While the cells positive for GFAP and nestin were similar between combination and BDNF-MSCs groups, they were higher than the HO-1- and GFP-MSCs groups (*P ≤ 0.05).

Nestin, as a differentiation and stem cells marker, was high only in the combination and BDNF-MSCs groups, at 13.07 ± 0.47% and 11.33 ± 3.23%, respectively. These values were significantly higher than those of the HO-1 (q = 6.45, *P ≤ 0.05) and GFP-MSCs groups (q = 6.45, *P ≤ 0.05), at 3.23 ± 1.50%, and 4.80 ± 3.10%, respectively (Figure 6(a,b)).

Histopathological Assessment

The injured spinal cord showed atrophy, fibrosis, and hemorrhages (Figure 7(a)). The degree of fibrosis was significantly the lowest in the combination group, compared to other groups (Figure 7(e)). This was followed by higher fibrosis in the BDNF and HO-1 groups, although lower compared to the GFP-MSCs (two-way ANOVA F = 10.23, ^*#P ≤ 0.05) (Figure 7(e)). Based on the visual examination, the combination group showed a well-organized parenchymal matrix with very few intervening fibrotic areas in the healthy area (Figure 7(c,d), arrowheads). The parenchymal matrix was comparatively less organized in the BDNF and HO-1-MSCs groups, with more intervening fibrotic areas in these groups than in the combination group. More robust fibrosis was observed in the GFP-MSCs group, with a more distorted parenchymal matrix (Figure 7(c,d)). More hemorrhagic areas were observed in the GFP-MSCs group than in the BDNF, HO-1, and combination groups (Figure 7(c,d), arrows).

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Figure 7.

Histopathology of spinal cord lesions stained with H&E stain. (a) An injured spinal cord showed fibrosis, atrophy, and hemorrhages. (b) Spinal cords sliced at the injured segment. (c) The fibrotic (red and pink) and healthy areas (purple) at the injured spinal cord section, at low magnification (40×), and (d) at high magnification (100×); scale bar 200 μm. (e) Percentage expression of fibrotic area. There is fibrosis at the epicenter that covers most of the healthy areas at the injured segment (c). The percentage fibrosis in the combination group was lower than in the other groups ((e), *P ≤ 0.05), followed by lower fibrosis in BDNF- and HO-1-MSCs groups compared to GFP-MSCs ((e), ^#P ≤ 0.05). Arrowheads denote fibrotic areas, while arrows indicate hemorrhages. The degree of hemorrhage was lower in combination, BDNF, and HO-1-MSCs groups compared with the GFP-MSCs group. Each picture (c) and bar (e) represents four samples per group (n = 4).