J Korean Neurosurg Soc.  2017 Jul;60(4):404-416. 10.3340/jkns.2016.1010.008.

Is There Additive Therapeutic Effect When GCSF Combined with Adipose-Derived Stem Cell in a Rat Model of Acute Spinal Cord Injury?

• Min J
• Kim JH
• Choi KH
• Yoon HH
• Jeon SR

• KMID: 2387878
• DOI: http://doi.org/10.3340/jkns.2016.1010.008

Abstract

OBJECTIVE
Functional and neural tissue recovery has been reported in many animal studies conducted with stem cells. However, the combined effect of cytokines and stem cells has not yet been adequately researched. Here, we analyzed the additive effects of granulocyte colony-stimulating factor (GCSF) on adipose-derived stem cells (ADSCs) infusion in the treatment of acute spinal cord injury (SCI) in rats.
METHODS
Four days after intrathecal infusion tubes implantation in Sprague-Dawley rats, SCI was induced with an infinite horizon impactor. In the Sham group (n=5), phosphate-buffered saline was injected 3, 7, and 14 days after SCI. GCSF, ADSCs, and ADSCs with GCSF were injected at the same time in the GCSF (n=8), ADSC (n=8), and ADSC+GCSF groups (n=7), respectively.
RESULTS
The ADSC and ADSC+GCSF groups, but not the GCSF group, showed significantly higher Basso-Beattie-Bresnahan scores than the Sham group during 8 weeks (p<0.01), but no significant difference between the ADSC and ADSC+GCSF groups. In the ladder rung test, all four groups were significantly different from each other, with the ADSC+GCSF group showing the best improvement (p<0.01). On immunofluorescent staining (GAP43, MAP2), western blotting (GAP43), and reverse transcription polymerase chain reaction (GAP43, nerve growth factor), the ADSC and ADSC+GCSF groups showed higher levels than the Sham and GCSF groups.
CONCLUSION
Our analyses suggest that the combination of GCSF and ADSCs infusions in acute SCI in the rat does not have a significant additive effect. Hence, when combination agents for SCI stem cell therapy are considered, molecules other than GCSF, or modifications to the methodology, should be investigated.

Keyword

Spinal cord injury; Mesenchymal stem cell; Combined modality therapy; GAP-43 protein; Granulocyte colony-stimulating factor

MeSH Terms

Animals
Blotting, Western
Combined Modality Therapy
Cytokines
GAP-43 Protein
Granulocyte Colony-Stimulating Factor
Mesenchymal Stromal Cells
Models, Animal*
Polymerase Chain Reaction
Rats*
Rats, Sprague-Dawley
Reverse Transcription
Spinal Cord Injuries*
Spinal Cord*
Stem Cells*
Cytokines
GAP-43 Protein
Granulocyte Colony-Stimulating Factor

Figure

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  Fig. 1 Diagram of intrathecal infusion catheter implantation.

• 

  Fig. 2 Flow cytometric histograms of rat ADSCs by FACScan flow cytometer (Beckton Dickson, San Jose, CA, USA). Adipogenic positive marker, CD73, and CD105 were expressed in 90.1% and 98.6%, respectively. Adipogenic negative marker, CD34, and CD45 were indicated as 3.8% and 0.6%, respectively. ADSC: adipose-derived stem cell.

• 

  Fig. 3 BBB locomotor scale test. The recovery of BBB locomotor function after SCI in rats treated by infusion with PBS (Sham group), injection with GCSF (GCSF group), infusion with ADSCs (ADSC group), or infusion with ADSCs and GCSF (ADSC+GCSF group). Black arrow is injection time point. *p<0.01. BBB: Basso-Beattie-Bresnahan, SCI: spinal cord injury, PBS: phosphate-buffered saline, GCSF: granulocyte colony-stimulating factor, ADSC: adipose-derived stem cell.

• 

  Fig. 4 Ladder rung test. The recovery of hindlimb function after SCI in rats treated by infusion with PBS (Sham group), injection with GCSF (GCSF group), infusion with ADSCs (ADSC group), or infusion with ADSCs and GCSF (ADSC+GCSF group). *p<0.01. SCI: spinal cord injury, PBS: phosphate-buffered saline, GCSF: granulocyte colony-stimulating factor, ADSC: adipose-derived stem cell.

• 

  Fig. 5 Histological assessment of axonal regeneration using a GAP43. A: Confocal microscopic pictures revealed that anti-GAP43 antibody staining in the spinal cord was greater in the ADSC and ADSC+GCSF groups than in the Sham or GCSF groups 8 weeks after SCI. B: Magnification of white arrow area from (A). Scale bar: 50 μm. ADSC: adipose-derived stem cell, GCSF: granulocyte colony-stimulating factor.

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  Fig. 6 Histological assessment of axonal regeneration using an MAP2. A: Confocal microscopic pictures revealed that anti-MAP2 antibody staining in the spinal cord was greater in the ADSC and ADSC+GCSF groups than in the Sham or GCSF groups 8 weeks after SCI. B: Magnification of white arrow area from (A). Scale bar: 50 μm. ADSC: adipose-derived stem cell, GCSF: granulocyte colony-stimulating factor, SCI: spinal cord injury, DAPI: 4′,6-diamidino-2′-phenylindole.

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  Fig. 7 Histological assessment of motor neuron marker using a ChAT. A: Confocal microscopic pictures revealed that anti-ChAT antibody staining in the spinal cord was greater in the ADSC+GCSF group than in other groups 8 weeks after SCI. B: Magnification of white arrow area from (A). Scale bar: 50 μm. GCSF: granulocyte colony-stimulating factor, ADSC: adipose-derived stem cell, SCI: spinal cord injury, DAPI: 4′,6-diamidino-2′-phenylindole.

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  Fig. 8 Western blots analysis of serotonin transporter. A: Injured epicenters of 0.5 cm spinal cord were extracted protein and analyzed by Western blots. B: When the band densities were converted into RDL value, the ADSC group showed significantly high density than Sham and GCSF groups. There was no statistical difference of RDL value between the ADSC and ADSC+GCSF groups. *p<0.05. RDL: relative density level, ADSC: adipose-derived stem cell, GCSF: granulocyte colony-stimulating factor.

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  Fig. 9 Western blots analysis of acetylcholine receptor. A: Injured epicenters of 0.5 cm spinal cord were extracted protein and analyzed by Western blots. B: When the band densities were converted into RDL value, the ADSC group showed significantly high density than other groups. *p<0.01. RDL: relative density level, ADSC: adipose-derived stem cell, GCSF: granulocyte colony-stimulating factor.

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  Fig. 10 Western blots analysis of GAP43. A: Injured epicenters of 0.5 cm spinal cord were extracted protein and analyzed by Western blots. B: When the band densities were converted into RDL value, the ADSC and ADSC+GCSF groups showed significantly high density than the Sham and GCSF groups. There was no statistical difference of RDL value between the ADSC and ADSC+GCSF groups. *p<0.01. RDL: relative density level, ADSC: adipose-derived stem cell, GCSF: granulocyte colony-stimulating factor.

• 

  Fig. 11 RT-PCR analysis of GAP43. A: Injured epicenters of 0.5 cm spinal cord were extracted mRNA and analyzed by RT-PCR. B: When the band densities were converted to RDL value, the ADSC and ADSC+GCSF groups showed significantly high density than the Sham and GCSF groups. The ADSC+GCSF group showed significantly higher density than ADSC group. *p<0.01. RT-PCR: reverse transcription polymerase chain reaction, RDL: relative density level, ADSC: adipose-derived stem cell, GCSF: granulocyte colony-stimulating factor.

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  Fig. 12 RT-PCR analysis of NGF. A: Injured epicenters of 0.5 cm spinal cord were extracted mRNA and analyzed by RT-PCR. B: When the band densities were converted into RDL value, the ADSC and ADSC+GCSF groups showed significantly high density than the Sham and GCSF groups. The ADSC+GCSF group showed significantly higher density than ADSC group. *p<0.01. RT-PCR: reverse transcription polymerase chain reaction, RDL: relative density level, ADSC: adipose-derived stem cell, GCSF: granulocyte colony-stimulating factor.

Reference

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