Abstract: BACKGROUND: Human umbilical cord mesenchymal stem cell-derived exosomes were found to be effective in promoting neural repair in spinal cord injury.
OBJECTIVE: To investigate whether exosomes derived from human umbilical cord mesenchymal stem cells are able to attenuate neuroinflammation and promote recovery of motor function by promoting polarization of microglia toward the M2 type. 
METHODS: Totally 48 SD rats were randomly divided into a sham operation group, a model group, and an exosome group (n=16 per group). A rat spinal cord injury model was established using the modified Allen method. The exosome group was injected with 20 μL of human umbilical cord mesenchymal stem cell-derived exosomes intrathecally via neuroendoscopy 24 hours after injury. At 3, 7, 14, and 21 days after modeling, the recovery of the motor function of the hind limbs of the rats was assessed by BBB scoring method combined with Rivlin’s slant plate test. The damage of spinal cord tissues was detected by using hematoxylin-eosin staining and Nissl staining. The expression levels of brain-derived neurotrophic factor and vascular endothelial growth factor A proteins were detected by western blot assay. The expression proportion of M1-type markers (inducible nitric oxide synthase) and M2 markers (arginase-1) in the spinal cord tissues was detected by immunofluorescence method. qRT-PCR and western blot assay were used to detect the expression levels of inducible nitric oxide synthase and arginase-1 in spinal cord tissues. ELISA was utilized to detect the levels of pro-inflammatory factors (tumor necrosis factor α, interleukin 1β, and interleukin 6) and anti-inflammatory factors (interleukin 10) levels in spinal cord tissues.
RESULTS AND CONCLUSION: (1) At 3, 7, and 14 days postoperatively, the BBB scores of the exosome group were better than those of the model group (P < 0.05). The angles of the Rivlin slanting plate experiments of the exosome group were significantly higher than those of the model group at 7 and 14 days postoperatively (P < 0.05). The results of hematoxylin-eosin staining and Nissl staining indicated that the spinal cord tissues and nerve injuries of the exosome group were reduced in comparison with those of the model group, and the levels of brain-derived neurotrophic factor and vascular endothelial growth factor A in spinal cord tissues of the exosome group were higher than those in the model group at 7 days postoperatively (P < 0.05). (2) Immunofluorescence experiments showed that the number of inducible nitric oxide synthase-positive microglial cells in the lesion area of the exosome group was significantly reduced and the level of Arg1-positive microglial cells increased in the lesion area of the exosome group compared with the model group at 7 days postoperatively (P < 0.05). qRT-PCR and western blot assay also confirmed the results of immunofluorescence experiments. (3) The secretion of pro-inflammatory factors tumor necrosis factor α, interleukin 1β, and interleukin 6 in spinal cord tissues of the exosome group was reduced compared with the model group (P < 0.05), whereas the secretion of the inflammation-suppressing factor interleukin 10 was increased compared with the model group (P < 0.05). These findings conclude that human umbilical cord mesenchymal stem cell-derived exosomes could promote the polarization of microglial cells from the M1 to the M2 type and decrease the release of pro-inflammatory factors, thereby reducing the secondary damage of neuroinflammation in spinal cord injury.

Key words: spinal cord injury, human umbilical cord mesenchymal stem cell, microglia, exosome, inflammation, neuroendoscopy, engineered stem cell