Lenti-BDNF Gene Therapy For Improving Inflammatory Microenvironment And Promoting Macrophage Polarization After Spinal Cord Injury

There is much evidence to suggest that brain-derived neurotrophic factor(BDNF) is a prominent candidate in promoting neuroprotection, axonal regeneration and synaptic plasticity following spinal cord injury(SCI). Although some evidence indicates that BDNF has potent anti-oxidative effects and may be involved in the regulation of the immune response, the effects of BDNF in the inflammatory response during the course of secondary damage after SCI is still unclear. The present study found that overexpressed BDNF at the lesion site promotes the inflammatory microenvironment including inhibit the expression of pro-inflammatory cytokines(IL-1β, TNF-a) and up-regulate the expression of anti-inflammatory cytokines(IL-10,IL-13) and promotes the shift of macrophage phnotype from M1 to M2. Moreover, the neuroprotective effect of BDNF can be observed with this injection method. Part one: The injection of Lenti-BDNF at the epicenter of the lesion site overexpressedsuccessfully and most of the infected cells are inflammatory cells.Materials and methods1.The construction of Lenti-BDNF vector; BDNF gene fragment was amplified by PCR, and then ligated with enzyme digested GV208 vector to construct recombinant vector GV208-BDNF. The positive clones were identified by PCR and DNA sequencing. Lentivirus carrying BDNF were packaged within 293 T cells by co-transfection of recombinant vector and lentivirus packaging system. The titer of lentivirus was determined by real-time PCR.2.SCI model in C57BL/6 mice; In order to choose the suitable SCI model for our study, two methods of SCI model were established including T10 dorsal hemisection and clip compression models.3.The injection of Lentivirus vectors and the identification of the infected cells; Lenti-EGFP and Lenti-BDNF vectors were injected in the epicenter of the lesion site with stereotaxic apparatus and Hamilton trace sampler. To confirm that what the population of EGFP cells were composed of, the sections were labeled with CD11 b and GFAP by immune-fluorescent staining.4.The total BDNF levels in the injured spinal cord segments; Four days after injection the expression of BDNF in the spinal cord segments containing the lesion site(1.5 mm rostral and 1.5 mm caudal to the lesion epicenter) were detected by ELISA.Results1.The DNA sequencing results showed that positive clones of GV208-BDNF vector was constructed successfully. The final virus titer was 2.00E+8TU/ml determined by real-time PCR.2.The T10 dorsal hemisection and clip compression models could damage the dorsal and lateral CST, All of the mice demonstrate a severe loss of locomotor ability of the hindlimb after the injury. The clip compression model could keep the intigraty of the dura mater and reduce the loss of the lentivirus vectors.3.Four days after injection(7 days after SCI), confocal microscopy of sagittal sections showed the EGFP+ cells were distributed in the epicenter of lesion site within lenti-EGFP and lenti-BDNF groups. In addition, confocal imaging showed that more than 80% of the lenti-virus infected cells were CD11 b positive cells.4.The total BDNF levels in the injured spinal cord segments were greatly increased in animals that received lenti-BDNF vector injections(62.63±8.74 ng/g) compared with those that received lenti-EGFP injections(0.59±0.15 ng/g) and those animals within the saline group(0.68±0.13 ng/g)(P<0.05).Conclusions1.GV208-BDNF lentivirus packaged successfully and could meet the needs of the experiments2.Clip compression model is more suitable for the injection of lentivirus vectors compared with dorsal hemisection model.3.Most of the lenti-virus infected cells were inflammatory cells.4.Result of ELISA confirmed that BDNF overexpressed successfully at the lesion site. Part 2: The overexpressed BDNF at the lesion site modulates the inflammatoryresponse and promotes the repair of injured spinal cord nerve injurySection 1: The overexpression of BDNF at the epicenter of lesion site promotes the shift of macrophage phenotype from M1 to M2 and modulates the inflammatory environmentMaterials and methods1.Adult female C57BL/6 mice were used. Animals were divided into groups based on the type of vectors received. Lentiviral vectors expressed either EGFP only(lenti –EGFP groups), or BDNF plus EGFP(lenti–BDNF groups). Animals in the control group were injected accordingly with saline(Saline group). Additionally, animals receiving T10 laminectomy without spinal cord injury were set up as a sham group.2.To investigate whether the continuous expression of BDNF at the lesion site could influence the macrophage phenotype, the expression profiles of macrophages were evaluated by immunofluorescence staining with CD16/32 and Arginase-1, at 7 and 14 days post-injury.3.To further characterize changes in macrophage polarization, we used flow cytometry to assess the M1 markers i NOS and CD16/32, as well as the M2 markers CD206 and Arginase-1, at 7 and 14 days post-injury.4.To examine whether the overexpressed BDNF could regulate the production of inflammatory cytokines in the injured spinal cord, we measured the levels of pro-inflammatory and anti-inflammatory cytokines such as IL-1β, TNF-α, IL-10 and IL13 by ELISA.5.BMDMs stimulated by BDNF were set to BDNF group in vitro experiment. To determine the effect of BDNF to the macrophage function and phenotype, the inflammatory cytokines were detected by q RT-PCR and the expression of arginase-1and i NOS were evaluated by western blot between the BDNF group and control group.Results1.Confocal imaging showed that higher numbers of arginase-1-positive cells were found in the lenti-BDNF group compared with the lenti-EGFP and saline groups. Furthermore, the number of CD16/32-positive cells was significantly reduced in animals that received lenti-BDNF injections compared to animals in the lenti-EGFP and saline groups.2.Phenotypic analysis of macrophages using flow cytometry showed that M1 markers i NOS and CD16/32 were significantly reduced in the lenti-BDNF group relative to lenti-EGFP and saline groups. On the contrary, M2 markers CD206 and Arginase-1 were significantly increased in animals that received lenti-BDNF injections compared with those in lenti-EGFP and saline groups(P<0.01).3.The production of IL-1β and TNF-α were significantly reduced in the animals that received lenti-BDNF vector injections relative to those in the lenti-EGFP and saline groups. Whereas, The levels of IL-10 and IL-13 were significantly elevated in the lenti-BDNF group relative to lenti-EGFP and saline groups.4.There was no significant difference between BDNF group and control group in the expression of inflammatory cytokines and the expression of arginase-1/i NOS associated with macrophage phenotype.Conclusions1.The overexpression of BDNF at the epicenter of lesion site promotes the shift of the macrophage phenotype from M1 to M2.2.The overexpressed BDNF inhibits the expression of pro-inflammatory cytokines and up-regulates the level of anti- inflammatory cytokines.3.BDNF has no directly effects on the function and phenotype of the BMDMs.As mentioned above, the overexpression of BDNF could modulate the local inflammatory response. Whereas, our data showed that BDNF has no directly effects on the function and phenotype of the macrophages in vitro experiment. Acquiring a better knowledge of the underlying mechanisms of these events needs further studies.Section 2: The overexpression of BDNF improves the repair of injured spinal cordMaterials and methods1.Motor function was assessed using the Basso Mouse Scale(BMS) open field test. The animals in each group(n=10) were tested at 1, 3, 7, 14, 21 and 28 days following injury.2.To evaluate the demyelination in the Lenti-EGFP and Lenti-BDNF groups, the sections were stained with luxol fast blue at 28 days post-injury.3.To trace the corticospinal tract(CST), tracer injections were performed at 14 days before being sacrificed.The dieback of the corticospinal tract could be quantified using Image Pro-Plus software.4.To observe the effect of BDNF on the injured nerve fibers, the average length of NF-200-positive nerve fibers at the lesion site was evaluated by immunofluorescence staining with NF-200 at 28 days post-injury.Results1.The time course of functional recovery was evaluated by the Basso Mouse Scale(BMS) score after SCI. In the open field test, significant locomotor recovery was observed in the lenti-BDNF group compared with the lenti-EGFP group.2.Luxol Fast Blue(LFB) staining showed marked demyelination in the lenti-EGFP group compared with the lenti-BDNF group.3.At 28 days post injury, significant CST axons retraction was observed in lenti-EGFP injected animals relative to lenti-BDNF group.4.The average lengthof NF-200-positive nerve fibers were significantly longer in the animals that received lenti-BDNF injections compared with the lenti-EGFP group.ConclusionsThe overexpression of BDNF at the epicenter of lesion site promotes the recovery of locomotor function and shows the obvious neuro-protective effects. However, we have to be aware that most of the lenti-virus infected cells were inflammatory cells, not the neurons, axons and oligodendrocytes. Accordingly, the roles of BDNF in immunity modulation may enhance neuroprotective effects and partially contribute to the locomotor functional recovery after SCI.