Human Brain-Derived Neurotrophic Factor Gene-Modified Bone Marrow Mesenchymal Stem Cells Combined With Erythropoietin Can Improve Acute Spinal Cord Injury.

OBJECTIVE: To assess the effect as well as mechanism of bone marrow mesenchymal stem cells (BMSCs) modified by the human brain-derived neurotrophic factor gene combined with erythropoietin (EPO) in the treatment of acute spinal cord injury (SCI) in rats.
METHODS: The Brain-derived neurotrophic factor (BDNF) gene was transected by a virus vector. Rats with SCI were randomly split into following groups: The normal saline (NS) group, the EPO group, The Basso, Beattie, and Bresnahan scores, messenger RNA BDNF expression, and apoptosis rates were compared between the 4 groups at 1, 3, 7, 14, and 21 days after SCI.
RESULTS: At 7, 14, and 21 days after operation, the expression of the BDNF gene in the other 3 groups was higher than that of the NS group, and the difference was statistically significant (P < .05). The apoptosis rate in the combined group was less than that of NS, EPO, and BDNF/BMSC groups, and the differences were statistically significant (P < .05).
CONCLUSION: Brain-derived neurotrophic factor gene-modified BMSC transplantation combined with EPO can promote the repair of nerve function after SCI in rats.

Introduction

The current methods to treat spinal cord injury (SCI) clinically include vertebral decompression and drug therapy.[1] However, these methods can only avoid secondary injury of the spinal cord; the lower limb sensation and motor function are not improved.[2] With recent advances in the understanding of and the research regarding the pathogenetic mechanism of SCI, using the migration ability of bone marrow mesenchymal stem cells (BMSCs) to protect the residual neuronal function provides a novel approach for the treatment on SCI.[3] Bone marrow mesenchymal stem cells differentiate toward the direction of neuronal axons under the influence of spinal cord microenvironment, and neurons can be regenerated and functionally reconstructed through this approach.[3,4]

However, a few problems remain unsolved, such as the stimulation of nerve growth factor that lacks stability and the ability of BMSCs for directional homing.[5] Brain-derived neurotrophic factor (BDNF) has been demonstrated to repair and regenerate the spinal cord tissue.[6] In some studies, BDNF was transferred into the receptor cell as a target gene and it could promote axon growth in the SCI area of rats and improve limb motor function.[7] Furthermore, erythropoietin (EPO) can mobilize BMSCs to the peripheral blood and enhance the proliferation and migration of BMSCs.[8] Thus, the purpose of the current study was to combine BDNF gene-modified BMSCs with EPO and thus, provide a novel, effective method to treat SCI.

Materials and Methods

Experimental Material and Equipment

Ninety adult sprague dawley (SD) rats, weighing 200 to 240 g, with an average body weight of 215 ± 10 g were used. Ten SD rats were used for the separation of BMSCs. All animals were provided by the Beijing animal experiment center. H293 cells were obtained from Procell Company (Wuhan, China). The Ad-GFP plasmid was purchased from (Invitrogen, Carlsbad, USA). Other materials and equipment used were as follows: carbon dioxide cell culture box (Heraeus Company, Germany), inverted phase contrast microscope CX23 (Olympus Company, Nikon, Japan), fluorescence microscope IX81 (Olympus Company), refrigerated centrifuge (Heraeus company), serum (Gibco Company, Jinan, China), Hanks solution (homemade), rat mesenchymal stem cells separating medium (Tianjing Haoyang Biological Technology Company, Tianjing, China), TUNEL apoptosis kit (Boshide Company, Beijing, China), and PrimeScript RT reagent Kit (RR037A, Takara Company, Dalian, China).

Culture of BMSCs

Rates were anesthetized by 1% pentobarbital. After being disinfected with 75% alcohol, the bilateral metaphysis were cut off, and the shafts were flushed with 5 mL of Hanks. This was followed by filtration through a 200-mesh cell strainer, and the shaft was blown and beaten into a single-cell suspension. The rat’s BMSCs separating medium was used for centrifugal separation, after which the separated cells were inoculated into a flasket. EDTA and trypsinization were used for the passage, separation, proliferation, and purification of overgrown cells.

Transfection of the BDNF genetic recombinant green fluorescent protein–adenovirus vector into the BMSCs Ad-GFP plasmid was linearized by Pac I, and the plasmid was then transfected into 293 cells by Lipofectamine TM2000. The transfected cells were collected in 7 to 10 days and frozen and thawed repeatedly to obtain the original virus, and a massive proliferation was done as described previously. The virus was purified by caesium chloride, and the virus titer was tested by doubling the dilution (plaque-forming unit, PFU). The messenger RNA (mRNA) sequence of BDNF was determined using the Genebank and was used for primer design, synthesis, and proliferation because the human-derived BDNF sequence was more accurate than that of rats; thus, the human-derived BDNF gene was selected by this study as the target gene. Next, the construction of pDC-BDNF-GFP was conducted, and the BDNF adenovirus vector pAdEasy-GFPCXCRGFP was successfully constructed. The AD MAX packaging system was used to pack and purify it.

The cultured pAdEasy-GFPCXCRGFP BDNF virus solution (experimental group) and pAdEasy-GFP virus solution (control group) were divided into 6 different titers including 1 × 105, 1 × 106, 1 × 107, 1 × 108, 1 × 109, and 1 × 1010 PFU/mL. The virus solution of each titer was added into 4 pores, and another 4 pores were set as controls. The virus solution was inoculated into BMSCs, and the cells were continually cultured at 37°C with 5% CO2 in an incubation box for 48 hours. The transfection rate was regularly detected, and the optimal virus titer was decided. The virus titer for this study was determined to be 2.45 × 1010 PFU/mL.

Construction of SCI Model and Grouping

Adult male SD rats were anesthetized by chloral hydrate, followed by fixation in the prone position. The spinal cord was exposed after being sanitized by 75% ethyl alcohol, and the SCI striker was used to establish the SCI model as has been described in the literature. A successfully established model was symbolized by hematomyelia and edema, wherein the rear limbs developed a flaccid paralysis and the tail developed a spastic swing. The rats were randomly divided into 4 groups using the method of the random number table: BMSCs with an equal density of 1 × 106/mL; the experimental group (combined group, N = 20), wherein 5000 U/kg EPO was administered before SCI, the BDNF gene was transfected into 5 μL of BMSCs (50 μL/min) that was injected into the injured spinal cord at its midpoint and 5 mm above and below the injured midpoint after the spinal cord had been injured; the pure EPO group (EPO group, n = 20), wherein 5000 U/kg EPO was administered before SCI, and an equivalent cell-free culture medium was injected into the spinal cord after it had been injured; the pure BDNF gene-transfected BMSCs group (BDNF/BMSC group, n = 20), wherein an equivalent dose of normal saline (NS) was administered through the abdominal cavity before SCI, and 20 μL of BDNF gene-transfected BMSCs (50 μL/min) was injected into the spinal cord after injury; and the control group (NS group, n = 20), in which equivalent NS was injected through the abdominal cavity 1 day before SCI, and an equivalent cell-free culture medium was administered to the injured spinal cord after SCI. This experiment was approved by the ethics committee of The Second Affiliated Hospital of Shandong First Medical University.

Basso, Beattie, and Bresnahan Scores

The motor function of the animals in the 4 groups was evaluated on days 1, 3, 7, 14, and 21 after transplantation. The evaluation criteria was based on the relatively sophisticated method of Basso, Beattie, and Bresnahan (BBB) scoring which provides a grade ranging from 0 to 21, as described in a previous report.[9] The rat’s abdomen was pressed at 10:00 to 12:00 hours to empty its bladder through urination. The animals were allowed to move freely on a smooth surface, and the movement of the bilateral posterior limbs of each animal and whether its gait was coordinated or not were independently observed, graded, and averaged by 2 observers according to the scale criteria. The higher the score, the better the motor function recovery of the rats.

Brain-Derived Neurotrophic Factor mRNA Expression

Following evaluation, in terms of neuromotor function, 4 rats from each group were injected with an overdose of chloral hydrate and killed. The spinal cord was dissected and placed into the RNA preserving fluid, which was stored in a −80°C refrigerator for further experimentation. The total RNA of the spinal cord was extracted using Trizol (Invitrogen). The ReverTra Ace qPCR reverse transcription kit was used to reversely transcribe the RNA into complementary DNA, based on the instructions provided on the kit. The target molecular primers were as follows: β-actin, forward 5′-GACAGTCAGCCGCATCTTCT-3′, forward 5′-GCGCCCAATACGACCAAATC-3; BDNF, forward: 5′-TCCCTGGCTGACACTTTT-3′, reverse: 5′-ATTGGGTAGTTCGGCATT-3′. The SYBR Green Realtime PCR Master Mix (Toyobo Co Ltd, Tokyo, Japan). Quantitative kit was used for quantitative real-time polymerase chain reaction. β-actin was used as an internal reference, and a relative expression of RNAs was calculated by 2−ΔΔCt method.

Apoptosis of Spinal Cord Tissue Detected by TUNEL

Four spinal cord tissue samples, including the ones at 1 cm above and below the injury region, were taken from each group and placed into 4% paraformaldehyde and fixed for 3 days, followed by gradient dehydration through phosphate buffer containing 20% and 30% sucrose. The tissue was then frozen and sectioned at −20°C. The specific procedures of TUNEL staining were performed according to the kit instructions. The nuclei of cells that were detected as positive by the TUNEL tissue apoptosis kit staining assumed a brown color. The internet platform provider (IPP) software was used to analyze the apoptosis rate of spinal cord tissue. Cell apoptosis rate (%) = apoptotic cell count/total microscopic cells × 100%.

Statistical Analysis

SPSS 20.0 was used for statistical analyses. Since the samples were detected in different times with same observation target. Thus, all data were multigroup and multi-time point observational data. Integral analysis was performed using a bifactor repeated measures variance analysis, based on the spherical test result. Pairwise comparisons were performed using the t test (intergroup) and the difference t test (inter-time point). The total test level α = .05 and the significance level of pairwise comparisons were adjusted, according to the Bonferroni correction method.

Results

Comparison of BBB Grading

The BBB grading data are listed in Table 1. Integral analysis (bifactor repeated measures variance analysis) revealed that the differences between groups, time points, and interaction between grouping and time were all statistically significant (P < .05); consequently, a pairwise comparison between groups and time points was conducted, the results of which are shown in Table 1, primarily revealing that on days 1, 3, 7, 14, and 21 after surgery, the BBB grading of the combined group was higher than that of the NS group, and the difference between both was statistically significant (P < .013). On days 1 and 3, the difference in scores between the NS group, the BDNF/BMSC group, and the EPO group was not statistically significant. On days 7, 14, and 21 after surgery, compared to the EPO group, the BDNF/BMSC group had a higher BBB grading, and the difference between both was statistically significant (P < .013).

Table 1.

Comparison of BBB Grading Between All Groups After SCI.a

	A: NS Group	B: BDNF/BMSC Group	C: EPO Group	D: Combined Group
T1: 1 day	3.21 ± 0.42	3.85 ± 0.38b	3.85 ± 0.31b	5.75 ± 0.39bcd
T2: 3 days	4.31 ± 0.66t	4.75 ± 0.85t	4.41 ± 0.38t	7.29 ± 0.59bcdt
T3: 7 days	6.14 ± 0.25t	7.62 ± 1.09bt	6.35 ± 0.51ct	8.74 ± 1.23bcdt
T4: 14 days	7.01 ± 0.89t	12.08 ± 1.14bt	7.45 ± 0.91ct	15.35 ± 1.22bcdt
T5: 21 days	8.75 ± 1.14t	14.26 ± 1.13bt	10.47 ± 1.49bct	19.39 ± 1.06bcdt
Integral analysis F, P	(Spherical test correction HF coefficient: 0.2943)
Intergroup comparison	200.769, 0.000
Inter-time point comparison	872.352, 0.000
Group × time point	278.095, 0.000

Abbreviations: BBB, Basso, Beattie, and Bresnahan; BDNF, brain-derived neurotrophic factor; BMSC, bone marrow mesenchymal stem cell; EPO, erythropoietin; HF, Huynh-Feldtepsilon; NS, normal saline; SCI, spinal cord injury.

a The significance marker for pairwise comparison in the table is P < α′, b, c, and d is, respectively, compared with A, B, and C; t is that P < α′ compared to T1 time point in each group; α′ = .013, which is calculated by the Bonferroni correction method.

Expression of BDNF mRNA

The data of expression of BDNF mRNA are listed in Table 2. Through integral analysis, the differences between groups, time points, and interactions between grouping and time were all observed to be statistically significant (P < .05); consequently, a pairwise comparison between groups and time points was continued, the results of which are shown in Table 2, mainly revealing that on day 1 after surgery, the combined group had a higher BDNF RNA than that in the other groups, and compared to the NS group, the difference was statistically significant (P < .013); on day 3 after surgery, the combined group and the BDNF/BMSC group had higher RNA levels than those in the NS group and the EPO group, and the difference was statistically significant (P < .013); on days 7, 14, and 21, the BDNF/BMSC, EPO, and combined groups had equally higher RNA levels than those in the NS group, and the differences were statistically significant (P < .013).

Table 2.

Postoperative Comparison of BDNF mRNA Expression Between All Groups.a,b

	A: NS Group	B: BDNF/BMSC Group	C: EPO Group	D: Combined Group
T1: 1 day	1.04 ± 0.07	1.05 ± 0.04	0.97 ± 0.06bc	1.51 ± 0.17bcd
T2: 3 days	0.56 ± 0.07t	1.74 ± 0.96bt	0.59 ± 0.26ct	3.28 ± 0.55bcdt
T3: 7 days	0.39 ± 0.17t	3.29 ± 1.26bt	1.34 ± 0.63bct	5.64 ± 1.14bcdt
T4: 14 days	0.33 ± 0.05t	3.54 ± 1.25bt	1.65 ± 0.69bct	8.24 ± 1.08bcdt
T5: 21 days	0.28 ± 0.05t	5.11 ± 0.53bt	2.52 ± 0.45bct	9.16 ± 1.66bcdt
Integral analysis F, P	(Spherical test correction HF coefficient: 0.3027)
Intergroup comparison	267.802, 0.000
Inter-time point comparison	561.459, 0.000
Group × time point	191.683, 0.000

Abbreviations: BDNF, brain-derived neurotrophic factor; BMSC, bone marrow mesenchymal stem cell; EPO, erythropoietin; HF, Huynh-Feldtepsilon; mRNA, messenger RNA; NS, normal saline.

a n = 20.

b The significance marker for pairwise comparison in the table is P < α′, b, c, and d is, respectively, compared with A, B, and C; t is that P < α′ compared to T1 time point in each group; α′ = .013, which is calculated by the Bonferroni correction method.

Comparison of Apoptosis Rate Between 4 Groups

The data regarding the apoptosis rate in the spinal cord is shown in Table 3. Integral analysis (bifactor repeated measures variance analysis) revealed that the differences between groups, time points, and interaction between grouping and time were all statistically significant (P < .05). Consequently, a pairwise comparison between groups and time points was conducted, the specific statistical results of which are listed in Table 3, mainly revealing that the apoptosis rate of each group reached its peak on day 7 after surgery; on day 1 after surgery, the difference in apoptosis rate of the spinal cord between all groups was not statistically significant (P > .05). On day 3, the combined group clearly had a lower apoptosis rate than that of the other 3 groups, and the difference was statistically significant (P < .013). However, the difference in apoptotic rate between the NS group and the BDNF/BMSC group was not statistically significant (P > .013). The EPO group had a lower apoptosis rate than that of the NS group, and the difference was statistically significant (P < .013). The apoptosis rate of each group on days 7, 14, and 21 after surgery is shown in Table 3. The apoptosis rate on day 21 after surgery was detected by a typical staining section, as shown in Figure 1.

Table 3.

Comparison of Spinal Apoptosis Rate (%) Between All Groups.a,b

	A: NS Group	B: BDNF/BMSC Group	C: EPO Group	D: Combined Group
T1: 1 day	11.04 ± 1.07	11.14 ± 1.04	12.97 ± 2.15bc	11.35 ± 1.17d
T2: 3 days	39.55 ± 2.06t	36.12 ± 3.96bt	32.57 ± 3.06bct	22.26 ± 3.54bcdt
T3: 7 days	35.42 ± 3.17t	33.25 ± 2.27bt	31.37 ± 1.62bct	18.65 ± 3.23bcdt
T4: 14 days	31.33 ± 1.05t	33.56 ± 2.26bt	22.66 ± 3.89bct	15.26 ± 3.18bcdt
T5: 21 days	24.30 ± 1.15t	25.09 ± 1.53t	16.50 ± 2.45bct	10.15 ± 2.66bcdt
Integral analysis F, P	(Spherical test correction HF coefficient: 0.4312)
Intergroup comparison	150.865, 0.000
Inter-time point comparison	2754.283, 0.000
Group × time point	139.483, 0.000

Abbreviations: BDNF, brain-derived neurotrophic factor; BMSC, bone marrow mesenchymal stem cell; EPO, erythropoietin; HF, Huynh-Feldtepsilon; NS, normal saline.

a n = 20.

b The significance marker for pairwise comparison in the table is P < α′, b, c, and d is, respectively, compared with A, B, and C; t is that P < α′ compared to T1 time point in each group; α′ = .013, which is calculated by the Bonferroni correction method.

Figure 1.

Stained section for test of cellular apoptosis rate for all groups on day 21 after surgery. A represents the NS group, B represents the BDNF/BMSC group, C represents the EPO group, and D represents the combined group; the black arrow represents the positive cell, ×20 times. BDNF indicates brain-derived neurotrophic factor; BMSC, bone marrow mesenchymal stem cell; EPO, erythropoietin; NS, normal saline.

Discussion

In this study, on days 1, 3, 7, 14, and 21 after SCI, the combined group had a higher BBB grading than the other 3 groups, and the difference was statistically significant (P < .05). The difference between the EPO group and the control group was not statistically significant, which indicated that the motor function deficit caused by a damaged spinal cord was improved by EPO combined with BDNF/BMSCs in the early and terminal stage of SCI, while pure EPO failed to improve the organism’s motor function after the SCI. Erythropoietin mobilizes BMSCs and promotes homing of the BMSCs to the site of injury in the spinal cord. Furthermore, EPO itself is able to reduce the inflammatory reaction at the site of injury and repair the injured endothelium.[10] However, the BMSCs that were transfected with the BDNF gene continuously expressed BDNF, which further promoted the repair of the injured spinal cord. Brain-derived neurotrophic factor is a basic protein extracted from the pig brain and is very important for the survival and growth of neurons.

A major strength of current study included following items: (1) We firstly combined human brain–derived neurotrophic factor (hBDNF) gene-modified BMSCs with EPO for treatment with SCI and found that EPO has synergistic effect with hBDNF gene-modified BMSCs for treatment with SCI, (2) final results shown that combined hBDNF gene-modified BMSCs with EPO was superior than hBDNF gene-modified BMSCs or EPO alone, and (3) combined hBDNF gene-modified BMSCs with EPO could prevent cord neurons from apoptosis.

Erythropoietin and BDNF have synergistic effect for preventing apoptosis of cord neurons. Wang et al[11] reported that treatment with EPO enhances the expression of BDNF in neural cell. Vinberg et al[12] conducted a randomized controlled trial and found that EPO could increase plasma BDNF levels in patients with affective disorders. Viviani et al[13] found that EPO protects primary hippocampal neurons through increasing the expression of BDNF. We summarized the relationship between EPO and BDNF in Figure 2. When compared with methylprednisolone, EPO seems to better increase the expression of PDGF-β and thus produce better function outcomes than methylprednisolone in SCI.[14] On the other hand, EPO could facilitate the recruitment of BMSCs to sites of SCI.[15]

Figure 2.

Schematic illustration of the effects of EPO and BDNF in preventing apoptosis of cord neurons. BDNF indicates brain-derived neurotrophic factor; EPO, erythropoietin.

BDNF initiates several signal transduction pathways after binding with the BDNF receptor, suppressing the apoptosis of neurons.[16] Moreover, BDNF can also effectively remove the intracellular activating oxide through the phosphatidylinositol-3-kinases/protein-serine-threonine kinase (PI3K/AKT) signaling pathway and hence exert an antiapoptotic effect on neurons.[17] Additionally, there are studies demonstrating that BMSCs have a curative effect on the removal of post-SCI active oxygen, which is also one of the reasons for its therapeutic effects.[18]

Furthermore, we confirmed through RNA levels that the expression of post-SCI BDNF is clearly elevated by the combination of BDNF genetically modified BMSCs and EPO. This is primarily because the BMSCs transfected by the BDNF gene through a viral vector can secrete high levels of the BDNF protein. The continuous secretion of BDNF may reduce the secondary injury caused by SCI and hence exert an endogenous neuroprotective effect. BMSCs secrete BDNF during the process of differentiation, and a synergistic effect between the cells and the protein allows BDNF secretion levels to reach a maximum.

Previous studies have suggested that SCI was primarily due to mechanical compression which leads to cell apoptosis. However, with increasing SCI research,[19] it was observed that an increased cell apoptosis rate caused by the process of autodestruction of multigene regulation is the primary cause of SCI.[20] It was concluded through the observation of spinal cell apoptosis rates at different time points that the maximal post-SCI cellular apoptosis rate was observed on day 7 after SCI, which was also when the secondary injury of SCI reached its maximum. The TUNEL staining results demonstrated that by the use of a combination of BMSCs that are genetically modified by BDNF with EPO, the cell apoptosis rate can be reduced to the maximum extent, the mechanism of which is as follows: (1) Antiapoptotic effect of BDNF: Akt molecules can be activated by the binding of highly expressed BDNF to the TrkB receptor, which in turn activates the expression of downstream antiapoptotic proteins and suppresses cell apoptosis[21]; (2) antiapoptosis effect of BMSCs: BMSCs can secrete the antiapoptosis protein during differentiation to suppress apoptosis of neurons and restore neuronal function; (3) EPO can suppress apoptosis of neurocytes following SCI by regulating anoxia and the expression of oxygen radicals. In summary, a combination of BMSCs that are genetically modified by BDNF with EPO can maximally suppress apoptosis of neurocytes following SCI and protect the functions of the remaining neurons.

To conclude, this research for the first time demonstrates the application of a combination of BDNF/BMSCs and EPO to treat SCI and discusses its therapeutic mechanism preliminarily. Finally, the combination of BDNF/BMSCs and EPO can exert a neuroprotective effect by increasing BDNF expression and suppressing the expression of spinal neuron, which is significantly effective in treatment with SCI. Clinical application awaits further study and discussion.