Effect of neuroglobin genetically modified bone marrow mesenchymal stem cells transplantation on spinal cord injury in rabbits

Abstract

Objective: This study aims to investigate the potentially protective effect of neuroglobin (Ngb) gene-modified bone marrow mesenchymal stem cells (BMSCs) on traumatic spinal cord injury (SCI) in rabbits.

Methods: A lentiviral vector containing an Ngb gene was constructed and used to deliver Ngb to BMSCs. Ngb gene-modified BMSCs were then injected at the SCI sites 24 hours after SCI. The motor functions of the rabbits were evaluated by the Basso-Beattie-Bresnahan rating scale. Fluorescence microscopy, quantitative real-time PCRs, Western blots, malondialdehyde (MDA) tests, and terminal deoxynucleotidyltransferase-mediated UTP end labeling assays were also performed.

Results: Ngb expression in the Ngb-BMSC group increased significantly. A more significant functional improvement was observed in the Ngb-BMSC group compared with those in the other groups. Traumatic SCI seemingly led to an increase in MDA level and number of apoptotic cells, which can be prevented by Ngb-BMSC treatment.

Conclusion: This study demonstrates that Ngb gene-modified BMSCs can strengthen the therapeutic benefits of BMSCs in reducing secondary damage and improving the neurological outcome after traumatic SCI. Therefore, the combined strategy of BMSC transplantation and Ngb gene therapy can be used to treat traumatic SCI.

Figure 1. Morphology and phenotypic characterization of cultured BMSCs.

(A) Primary BMSCs at 5 days of culture (magnification: ×100). (B) BMSCs at passage 3 (magnification: ×100). (C) Flow cytometric analysis of cultured BMSCs with CD29, CD34, CD45, and CD90 antibodies. [Bar  = 500 µm (A, B)].

Figure 2. Transduction of Ngb into BMSCs with LV-Ngb.

(A, B) eGFP expression in BMSCs 6 days after Ngb – eGFP transduction (magnification: ×100). (A) Phase contrast. (B) Fluorescence microscopy. (C) Western blot detection of Ngb protein expression (lane 1, before transduction; lane 2, 3 days after transduction; lane 3, 6 days after transduction) [Bar  = 500 µm (A, B)].

Figure 3. Fluorescence microscopy of the injured spinal cords.

eGFP fluorescence detected with a fluorescence microscope on days 7 (A), 14 (B), and 21 (C) after SCI (magnification, ×200) [Bar  = 200 µm (A, B,C)].

Figure 4. Ngb mRNA expression in the injured spinal cords on days 3, 7, 14, and 21 after SCI in each group.

The data were plotted as mean ± SD (n = 6). ^* P<0.05 versus the Control and the NS groups (one-way ANOVA), ^# P<0.05 versus the BMSC group (one-way ANOVA).

Figure 5. Detection of Ngb protein in the spinal cords.

(A) Ngb and β-actin detected by Western blot in the spinal cord lysates obtained from four groups at four different time points. The blots are representative examples from six experiments per group per time point. (B) The quantitative mean ± SD data shows the level of Ngb protein expression. The band density is normalized as the ratio of Ngb: β-actin (n = 6). ^* P<0.05 versus the Control and the NS groups (one-way ANOVA), ^* P<0.05 versus the BMSC group (one-way ANOVA).

Figure 6. Hindlimb functional assessment with the BBB rating scale on days 1, 3, 7, 14, and 21 after SCI in each group.

The data are plotted as mean ± SD. ^* P<0.05 versus the Control and the NS groups (repeated-measures ANOVA), ^* P<0.05 versus the BMSC group (repeated-measures ANOVA).

Figure 7. MDA levels in the injured spinal cords in the four groups at four different time points.

Data were plotted as mean ± SD (n = 6). ^* P<0.05 versus the Control and the NS groups (one-way ANOVA), ^# P<0.05 versus the BMSC group (one-way ANOVA).

Figure 8. Apoptotic indexes at four different time points in each group.

The data are plotted as mean ± SD (n = 6). ^* P<0.05 versus the Control and the NS groups (one-way ANOVA), ^# P<0.05 versus the BMSC group (one-way ANOVA).

Figure 9. Apoptosis in the injured spinal cords on day 7 after SCI (magnification, ×400).

(A) Control; (B) NS; (C) BMSC; (D) Ngb-BMSC groups. The TUNEL positive cells were stained dark brown. The number of positive cells was significantly lower in the Ngb-BMSC group compared with the numbers in the Control, NS, and BMSC groups [n = 6, Bar  = 100 µm (A, B, C, D)].