J Vet Sci.  2017 Sep;18(3):377-386. 10.4142/jvs.2017.18.3.377.

Effect of canine mesenchymal stromal cells overexpressing heme oxygenase-1 in spinal cord injury

• Lee SH
• Kim Y
• Rhew D
• Kim A
• Jo KR
• Yoon Y
• Choi KU
• Jung T
• Kim WH
• Kweon OK

• KMID: 2412456
• DOI: http://doi.org/10.4142/jvs.2017.18.3.377

Abstract

Heme oxygenase-1 (HO-1) is a stress-responsive enzyme that modulates the immune response and oxidative stress associated with spinal cord injury (SCI). This study aimed to investigate neuronal regeneration via transplantation of mesenchymal stromal cells (MSCs) overexpressing HO-1. Canine MSCs overexpressing HO-1 were generated by using a lentivirus packaging protocol. Eight beagle dogs with experimentally-induced SCI were divided into GFP-labeled MSC (MSC-GFP) and HO-1-overexpressing MSC (MSC-HO-1) groups. MSCs (1 × 10⁷ cells) were transplanted at 1 week after SCI. Spinal cords were harvested 8 weeks after transplantation, after which histopathological, immunofluorescence, and western blot analyses were performed. The MSC-HO-1 group showed significantly improved functional recovery at 7 weeks after transplantation. Histopathological results showed fibrotic changes and microglial cell infiltration were significantly decreased in the MSC-HO-1 group. Immunohistochemical (IHC) results showed significantly increased expression levels of HO-1 and neuronal markers in the MSC-HO-1 group. Western blot results showed significantly decreased expression of tumor necrosis factor-alpha, interleukin-6, cycloogygenase 2, phosphorylated-signal transducer and activator of transcription 3, and galactosylceramidase in the MSC-HO-1 group, while expression levels of glial fibrillary acidic protein, β3-tubulin, neurofilament medium, and neuronal nuclear antigen were similar to those observed in IHC results. Our results demonstrate that functional recovery after SCI can be promoted to a greater extent by transplantation of HO-1-overexpressing MSCs than by normal MSCs.

Keyword

heme oxygenase-1; mesenchymal stromal cells; spinal cord injuries

MeSH Terms

Animals
Blotting, Western/veterinary
Dogs/*injuries/metabolism
Female
Heme Oxygenase-1/*metabolism
Mesenchymal Stromal Cells/*enzymology/metabolism
Spinal Cord/metabolism/pathology
Spinal Cord Injuries/metabolism/pathology/*veterinary
Heme Oxygenase-1

Figure

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  Fig. 1 Fluorescence microscopy images and cell viability results. (A) Green fluorescence protein (GFP) overexpressing cADMSCs. Top panels, cADMSC; middle panels, GFP-labeled cADMSCs (MSC-GFP); lower panels, GFP-labeled HO-1 overexpressing cADMSCs (MSC-HO-1). GFP expression was identified in MSC-GFP and MSC-HO-1 via fluorescence microscopy. Right panels are higher magnification images of left panels, respectively. (B) Cell viability of the three cell types showing no significant difference. Scale bars = 50 µm.

• 

  Fig. 2 Protein expression in cADMSCs overexpressing heme oxygenase 1 (HO-1) and their antioxidant effects. (A) Western blot results for cADMSCs, MSC-GFP, and MSC-HO-1. Expression of HO-1 in MSC-HO-1 was significantly higher than it was in the cADMSCs and MSC-GFP, and the MSC-GFP and MSC-HO-1 showed a significant increase in GFP expression relative to that in cADMSCs, but there was no difference between overexpressed cells (*p < 0.05 vs. cADMSCs, †p < 0.05 vs. MSC-GFP). (B) Antioxidant capacity of cADMSCs, MSC-GFP, and MSC-HO-1. Uric acid equivalent level of MSC-HO-1 was significantly higher than that of the other groups. There was no difference between the cADMSCs and MSC-GFP (*p < 0.05 vs. PBS, †p < 0.05 vs. cADMSCs, and ‡p < 0.05 vs. MSC-GFP).

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  Fig. 3 Behavioral analysis results using Basso, Beattie, and Bresnahan (BBB) scores and Tarlov scales. (A) BBB scores before and during the 8 weeks after transplantation. Post-transplantation, the BBB scores of the MSC-HO-1 group were significantly improved at 7 and 8 weeks after transplantation compared with the GFP-labeled cADMSCs (MSC-GFP) scores (*p < 0.05). Hindlimb locomotion at 8 weeks after transplantation were determined by two grading system: (B) revised Tarlov scales and (C) modified Tarlov scales. Higher motor function recovery was indicated in the MSC-HO-1 group than the MSC-GFP group (*p < 0.05).

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  Fig. 4 Histopathological analysis with H&E staining. Histological analysis of spinal cord lesions stained with H&E. (A) MSC-GFP group; upon magnification of the injury epicenter, fibroblast-like cell proliferation was detected. (B) MSC-HO-1 group; spinal cord lesion was limited to the compression region and a greater reduction in fibrotic changes was observed compared with those in the MSC-GFP group. Also, upon magnification, a reduction in fibroblast-like cell proliferation was observed. (C) Quantification of fibrotic tissue was showed significant difference between groups (*p < 0.05). 40× (top panels of A and B), 200× (bottom panels of A and B; enlargements of squares of the middle panels).

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  Fig. 5 Immunohistochemical assessments. (A) Immunofluorescence staining at 8 weeks after transplantation. Injured spinal cord lesions were immunostained for heme oxygenase 1 (HO-1), glial fibrillary acidic protein (GFAP), β3-tubulin, neurofilament M (NF-M), neuronal nuclear antigen (NeuN), galactosylceramidase (GALC), and phosphorylated-signal transducer and activator of transcription 3 (p-STAT3; red); transplanted cells (green fluorescent protein [GFP]); each nucleus was stained with DAPI (blue). (B), (C), and (D) showed quantification of immunostaining results. (B) Expression of red positive markers of antibodies. Expressions of the HO-1, β3-tubulin, NF-M, and NeuN were higher in the MSC-HO-1 group than MSC-GFP group, and expression of GFAP was adversely affected. The number of transplanted cells expressing GFP was not different between the two groups (*p < 0.05). (C) GFP expression shows no difference between the groups. (D) Comparison of the origins of HO-1 and neural cells in the injured site, whether from implanted MSCs or endogenous host cells. The expression of most neural cell markers from endogenous origins (not GFP labeled) were significantly higher than those of transplanted cells origins (GFP labeled) in both groups (*p < 0.05). However, expression rate of HO-1 marker from transplanted cells origins was significantly higher than that from endogenous origin cells in the MSC-HO-1 group (*p < 0.05). Scale bars = 50 µm.

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  Fig. 6 Western blot analysis. Protein expressions at 8 weeks after transplantation were evaluated by western blotting. Overexpression markers, heme oxygenase 1 (HO-1) and green fluorescent protein (GFP); inflammatory markers, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), cycloogygenase 2 (COX2), phosphorylated-signal transducer and activator of transcription 3 (p-STAT3), and GALC; neuronal markers, glial fibrillary acidic protein (GFAP), β3-tubulin, NF-M, and neuronal nuclear antigen (NeuN). Data presented as means ± SE of three independent experiments. The graph and bars depict means ± SE of four dogs per group, as determined by observed densitometry relative to β-actin (*p < 0.05, †p < 0.001).

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