Effect Of M2 Macrophages Adoptive Transfer On Locomotor Functional Recovery In Adult Rats After Spinal Cord Injury

Background: A variety of immune cell types infiltrate into the local microenvironment at the site of spinal cord injury, such as lymphocytes, mononuclear macrophages.A previous study found that macrophages may play an important role in tissue repair after spinal cord injury. After spinal cord injury, a large number of classically activated pro-inflammatory macrophages(M1) and alternatively activated anti-inflammatory macrophages(M2) appeared around injured site. M1 macrophages are neurotoxic, while M2 macrophages promote axonal regeneration after central nervous system(CNS) injury, Moreover, the M1 response is rapidly induced and then maintained, whereas M2 induction is transient, Therefore, increasing the M2 cell population and prolonging the presence of this macrophage subtype in the injured local microenvironment may represent a promising strategy for tissue repair after SCI.Objective: To investigate effect of M2 macrophages adoptive transfer on locomotor functional recovery in adult rats after spinal cord injury and to explore its mechnism.Method:(1) After cultured in L929-conditioned medium for approximately 7-day, bone marrow cells derived from rat leg bones were differentiated into macrophages which were defined as M0. Then M0 macrophages were induced to polarize into M1 or M2 macrophages under different conditions.(2) The specific markers of M0, M1 and M2 macrophages were detected by immunocytochemistry or flow cytometry to identify their purity.(3) The levels of IL-6, NO, IL-10, TNF-a and TGF-b in culture supernatants of M0, M1 and M2 macrophages were determined by enzyme linked immuosorbent assay(ELISA).(4) Rat model of contusive SCI was established using a New York University impactor. At the 7th day after SCI, CFSE-labeled M0, M1 and M2 macrophages were adoptive transferred into model rats, respectively.(5) The expressions of IL-4,-10,-13,-6, i NOS and TNF-g m RNAs in the injury spinal cord were detected by real-time PCR.(6) The infiltration and portion of transferred M0, M1 and M2 macrophages in injured spinal cord was detected using fluorescence microscope and flow cytometry 7 days after cell transfer.(7) The portion of Th1/Th2 cells in injured spinal cord was detected by immunocytochemistry 7 days after cell transfer.(8) Basso, Beattie and Bresnahan(BBB) locomotor rating scale, footprint analysis, grid walk were used to observe the effect of cell-transfer on motor function recovery.(9) Six weeks after SCI, animals were sacrificed and the spinal cords were blocked into 10 mm segments. The effects of cell-transfer on lesion volume, neuron protection, myelin preservation and remylination were observed using Neutral Red staining, Luxol fast blue(LFB) staining, Toluidine blue staining and electron microscopy, respectively.Results:(1) The M0, M1 and M2 macrophages were successfully isolated and cultured; the purity of cells met requirement for this experiment.(2) The result of ELISA showed that IL-6, NO and TNF-? levels were markedly higher in M1 than M0 and M2 in cell culture supernatants; whereas M2 cells produced higher levels of IL-10 and TGF-? than the other macrophage subtypes.(3) Seven days after cell transfer,the numbers of CD68+ cell were similar in vehicle, M0, M1 and M2 celltransferred groups(1096 ±395, 1462 ±548, 1687 ± 540 and 1585 ± 560 per mm2, respectively). However, there was less M1 cells(CD68+CCR7+) in the M2 celltransfer group, but more M2 cells(CD68+Arg1+) were observed in the M2 celltransfer group as compared to the other three groups. There were no significant difference observed among the other three groups.(4) The M2 cell-transfer was conducive to M2 polarization from endogenous macrophages and resident microglia. The numbers of M2 cells in vehicle, M0, M1 and M2 cell-transferred groups were 38 ± 11, 40 ± 10, 35 ± 12 and 512 ± 106/mm2, respectively.(5) The M2 cell-transfer induced Th2 cell polarization of Th cells. The numbers of Th2 cells were 38 ± 13, 36 ± 15, 41 ± 16 and 540 ± 146/mm2 in vehicle, M0, M1 and M2 cell-trasferred groups, respectively.(6) Real-time RT-PCR showed that there was lower IFN-? and higher IL-10,-13 m RNA expressions in M2 cell-transferred group as compared to the other three groups. There were higher IL-6, TNF-? and i NOS m RNA expression in M1 cell-transferred group as compared to the other three groups.(7) There were higher BBB scores, footprint analysis scores and fewer footfall errors in M2 cell-transferred group as compared to the other three groups.(8) There was smaller lesion volume, larger volume of residual myelination and higher number of ventral horn motor neurons in M2 cell-transferred group as compared to the other three groups.Conclusion: Adoptive transfer of M2 macrophages results in histological improvement and locomotor functional recovery in adult rats with SCI. M2 cells induce Th2 cell polarization by producing anti-inflammatory cytokines, which in turn produce anti-inflammatory cytokines that promote M2 cell polarization, creating a microenvironment that promotes tissue repair.