Allogenic Adipose Derived Stem Cells Transplantation Improved Sciatic Nerve Regeneration in Rats: Autologous Nerve Graft Model

Abstract

We examined the effect of transplantation of allogenic adipose-derived stem cells (ADSCs) with properties of mesenchymal stem cells (MSCs) on posttraumatic sciatic nerve regeneration in rats. We suggested an approach to rat sciatic nerve reconstruction using the nerve from the other leg as a graft. The comparison was that of a critical 10 mm nerve defect repaired by means of autologous nerve grafting versus an identical lesion on the contralateral side. In this experimental model, the same animal acts simultaneously as a test model, and control. Regeneration of the left nerve was enhanced by the use of ADSCs, whereas the right nerve healed under natural conditions. Thus the effects of individual differences were excluded and a result closer to clinical practice obtained. We observed significant destructive changes in the sciatic nerve tissue after surgery which resulted in the formation of combined contractures in knee and ankle joints of both limbs and neurotrophic ulcers only on the right limb. The stimulation of regeneration by ADSCs increased the survival of spinal L5 ganglia neurons by 26.4%, improved sciatic nerve vascularization by 35.68% and increased the number of myelin fibers in the distal nerve by 41.87%. Moreover, we have demonstrated that S100, PMP2, and PMP22 gene expression levels are suppressed in response to trauma as compared to intact animals. We have shown that ADSC-based therapy contributes to significant improvement in the regeneration.

Keywords: DRG; IVIS Spectrum; PCR; PNI; autologous nerve graft; myelin fibers.

FIGURE 1

Autologous rat sciatic nerve graft. (A) Open wounds to sciatic nerves. (B) Autologous left 10 mm, sciatic nerve grafting using the right sciatic nerve fragment stimulated by ADSCs. (C) Autologous right 10 mm, sciatic nerve grafting using the left sciatic nerve fragment without additional cells.

FIGURE 2

Expression of CD 29, CD 44, STRO, Thy-1 by undifferentiated ADSCs subpopulations. Row 1: Confocal analysis of the expression of CD 29 (A), e-GFP (B), DAPI (C), Merge (D). Row 2: Confocal analysis of the expression of CD 44 (E), e-GFP (F), DAPI (G), Merge (H). Row 3: Confocal analysis of the expression of STRO (I), e-GFP (J), DAPI (K), Merge (L). Row 4: Confocal analysis of the expression of Thy-1 (M), e-GFP (N), DAPI (O), Merge (P). Bar: 100 μm.

FIGURE 3

Lifetime assessment survival and migration of eGFP-ADSCs using the IVIS Spectrum system. Green fluorescence indicates the ADSCs survival and migration on the experimental side (left side) (A). There is no fluorescence on the contralateral control side (right side) (B) 14 days after the operation.

FIGURE 4

Neurons of L5 spinal ganglion. (A) Neurons of L5 left spinal ganglion, (B) neurons of L5 right spinal ganglion. Top: histological staining with azure-eosin. Bar: 200 μm. (C) Number of surviving neurons of L5 spinal ganglia: 1792.8 ± 58.8 (Intact, n = 6), 1166.1 ± 142.3 (L5 left, Experimental, n = 5) and 858.3 ± 66.2 (L5 right, Control, n = 5). Bottom: quantification of survived neurons of L5 spinal ganglia. Error bars represent standard error mean. Differences were statistically significant between the groups (^∗), (^∗P < 0.05, one-way ANOVA, Tukey’s test).

FIGURE 5

Vascularization 30 and 60 days after the trauma. The distal segment of the sciatic nerve vascularization 30 and 60 days after the trauma. Bottom: apu (absolute perfusion units): 9.92 ± 1.04 (Intact, n = 6), 10.18 ± 1.4 (PN left – 30 days, n = 8), 7.12 ± 1.06 (PN right – 30 days, n = 8), 8.19 ± 1.16 (PN left – 60 days, n = 8), 6.32 ± 1.84 (PN right – 60 days, n = 8). Error bars represent standard error mean. Differences were statistically significant between the left sciatic nerve (experimental side) and the right sciatic nerve (control side) (^∗), (^∗P < 0.05, one-way ANOVA, Tukey’s test).

FIGURE 6

Myelin fibers of the distal segment of the sciatic nerve. (A) Myelin fibers of the distal segment of the left sciatic nerve, (B) Myelin fibers of the distal segment of the right sciatic nerve. Top: histological staining with osmium and methylene blue. Bar: 50 μm. (C) Number of myelinated fibers: 440.67 ± 29.06 (Intact, n = 6), 210.17 ± 26.55 (PN left, n = 5), 122.17 ± 12.61 (PN right, n = 5). Bottom: quantification of myelin fibers of the sciatic nerve. Error bars represent standard error mean. Differences were statistically significant between the groups (^∗), (^∗P < 0.05, one-way ANOVA, Tukey’s test).

FIGURE 7

The transmission electron microscopic examination of the distal segment of the right and left sciatic nerve. (A) Tissue samples with insert (control group): m- myelin fibers, ^∗ – nuclei of Schwann cells, light background – connective tissue, c – cells with signs of degeneration, m – myelin fibers. Bar: 5 μm. (B) Tissue of the left sciatic nerve samples: m – myelin fibers, ^∗ – nuclei of Schwann cells, pointer – bundles of myelinated and unmyelinated fibers, n – cell nuclei. Bar: 5 μm.

FIGURE 8

S100, PMP22, and PMP2 mRNA expression in the left (with ADSCs transplantation) and right (without ADSCs transplantation) spinal ganglia (A–C) and the sciatic nerves (D–F). Differences were statistically significant between the groups (^∗). ^∗P < 0.05, ^∗∗P < 0.01, one-way ANOVA, Tukey’s test.