Effects of hypothermia combined with neural stem cell transplantation on recovery of neurological function in rats with spinal cord injury

Abstract

The microenvironment of the injured spinal cord is hypothesized to be involved in driving the differentiation and survival of engrafted neural stem cells (NSCs). Hypothermia is known to improve the microenvironment of the injured spinal cord in a number of ways. To investigate the effect of NSC transplantation in combination with hypothermia on the recovery of rat spinal cord injury, 60 Sprague‑Dawley female rats were used to establish a spinal cord hemisection model. They were divided randomly into three groups: A, spinal cord injury group; B, NSC transplantation group; and C, NSC transplantation + hypothermia group. At 1, 2, 4, 6 and 8 weeks post‑injury, the motor function of all animals was evaluated using the Basso, Beattie and Besnaham locomotor scoring system and the inclined plane test. At 4 weeks post‑transplantation, histological analysis and immunocytochemistry were performed. At 8 weeks post‑transplantation, horseradish peroxidase nerve tracing and transmission electron microscopy were conducted to observe axonal regeneration. The outcome of hind limb motor function recovery in group C significantly surpassed that in group B at 4 weeks post‑injury (P<0.05). Recovery was also observed in group A, but to a lesser degree. For the pathological sections no neural axonal were observed in group A. A few axon‑like structures were observed in group B and more in group C. Horseradish peroxidase‑labeled neurofibers and bromodeoxyuridine‑positive cells were observed in the spinal cords of group C. Fewer of these cells were found in group B and fewer still in group A. The differences among the three groups were significant (P<0.05). Using transmission electron microscopy, newly formed nerve fibers and myelinated nerve fibers were observed in the central transverse plane in groups B and C, although these nerve fibers were not evident in group A. In conclusion, NSC transplantation promoted the recovery of hind limb function in rats, and combination treatment with hypothermia produced synergistic effects.

Figure 1

Morphology of neural stem cells. (A) Rat mesenchymal stem cells were cultured for 2 days. Adherent cells extended and became spindle-shaped. (B) Rat mesenchymal stem cells were cultured for 7 days and grown around a clone. (C) The 3rd passage of mesenchymal stem cells fused together and became arranged in a bunched or radiating shape. Scale bar, 50 μm.

Figure 2

BBB scores and incline plate test. (A) BBB scores and (B) Tilt angles of each group at 1, 2, 4, 6 and 8 weeks following spinal cord injury. BBB, Basso, Beattie and Bresnaham locomotor scoring system. Significance levels are as shown in Tables I and II.

Figure 3

Histological analysis using H&E staining. (A) Four weeks post-injury, in group A at the affected site of the damaged spinal cord exhibited a clear cavity formation. (B) In group B, astrocytes congregated at the edge of the affected site and formed scars at the junction of complete spinal cord and damaged spinal cord. The tissue cavity was smaller than that in group A but larger than that in group C. (C) In group C, astrocytes underwent a reactive hypertrophy, and congregated and formed scars at the edge of the affected site, resulting in the cavity disappearing. Arrows show Syringomyelia and scar healing. Scale bar, 100 μm.

Figure 4

Immunohistochemical staining showing the number of BrdU-positive cells in the SCI lesion tissues in rats in (A) Group A, (B) group B and (C) group C. (D) Graph displaying quantity of BrdU-positive cells in each group. By analysis of variance and Dunnett’s t-test comparison, the number of BrdU-positive cells in group C was found to be increased compared with group B (P<0.05) and group A (P<0.01). Arrows show BrdU-positive cells. Scale bar, 50 μm. BrDU, bromodeoxyuridine; SCI, spinal cord injury.

Figure 5

HRP retrograde nerve tracing. (A) In group A, few HRP-positive granule-labeled nerve fibers were observed. (B) In group B, fewer HRP-positive nerve fibers were observed than in group C, but more than group A. (C) In group C, a large quantity of HRP-positive granule-labeled nerve fibers were observed. (D) Graph displaying the number of HRP-positive cells in each group. The number of HRP-positive nerve fiber bundles in rat SCI tissues exhibited significant differences among the three groups at 8 weeks post-injury (P<0.01). Arrows show HRP-positive granule-labeled nerve fibers. Scale bar, 50 μm. HRP, horseradish peroxidase; SCI, spinal cord injury.

Figure 6

Transmission electron microscopy. (A) A glial scar and a small number of myelinated nerve fibers are shown in group A. (B) The number of myelinated nerve fibers and non-myelinated nerve fibers at the injury site in group B was greater than that in group A but less than that in group C. (C) Numerous myelinated and non-myelinated nerve fibers were observed in group C, with more axons and intact myelin. Arrows show a glial scar and a small number of myelinated nerve fibers. Scale bar, 0.1 μm.