Vaccination With Dendritic Cells Pulsed With Homogenate Protein Of Spinal Cord Promotes Functional Recovery From Spinal Cord Injury In Mice

Spinal cord injury (SCI) is a major medical problem worldwide, usually causing devastating outcome, and realistic goals of functional recovery have only recently been acknowledged. Studies have indicated that protective autoimmunity, a properly controlled T cells-mediated, antigen-specific immunity in SCI, plays a pivotal role in regrowth of the injured spinal cord and its protection from secondary degeneration. Studies show that passive or active immunization with T-cells specific to CNS-associated myelin antigens, or local implantation of macrophages activated by an autologous sciatic nerve reduces secondary degeneration and improves functional recovery in rat and mouse models of optic nerve crush or spinal cord contusion.Dendritic cells (DCs) are antigen presenting cells (APCs). Study indicates that vaccination locally with DCs pulsed with myelin basic protein (MBP) or peptides derived from MBP can improve the recovery from SCI in rat. However, the facts that the difficulty in harvesting MBP, and the purity in constituent activating single immune reaction, and the trauma of local injection, limit the use of vaccination. Homogenate of spinal cord contains all kinds of the autoimmune antigens theoretically. Could vaccination with DCs pulsed with homogenate of spinal cord (hpDCs) improve functional recovery from SCI? Could injection of cells peritoneally have same effect as locally does? To find out the answers, in the present study we injected immature DCs pulsed with homogenate of spinal cord (hpDCs) or pure homogenate of spinal cord (hp), or pure immature DCs, locally or peritoneally, into the site of spinal cord contusion in mice to seek the results of the above presumption.Materials and methods:1. Spinal cord injury in mouse.Adult BALB/c mouse was used. The spinal cord was severely contused in T10 vertebrate, caused by microsurgery artery clip. 2. Immature DCs, hp and hpDCs were harvested.3. Vaccination. Three groups were established, injected DCs, hp, or hpDCs, respectively, 24h after SCI. Each group was divided into two subgroups, according to the way of administration was local or peritoneal.4. Measurement of neural functional recovery. BBB open-field locomotor testing was undertaken on 28, 56 and 84 days post injection (dpi).5. HE stain.Spinal cords were harvested on 28, 56 and 84dpi, and HE stain was processed to observe the pathological changes.6. Immunohistochemistry (IHC) and immunohistochemcial fluorescene stain: Assess the expression of glial fibrillary acidic protein (GFAP), Nestin and neurofilament (NF) and measure the areas of cysts and thickness of glial scar in the injuryed spinal cord.7. To observe the correlation between glial scar and axons around the cyst, biotinylated dextran amine (BDA) antegradation or horseradish peroxidase (HRP) retrogradation and IHC double stain were processed on 84dpi.Results:1. BBB score declined to 0 after SCI, then recovered slowly, and reached a highest plateau (14.0±2.0) on 28dpi in hpDCs group, while reached lower plateaus at 35dpi in DCs and hp groups(10.0±2.0,10.4±1.8, respectively). Difference was significant between hpDCs group and another two groups (p<0.01), while no differences were observed between DCs and hp group, or between mice injected locally and peritoneally.2. Glial scars and cysts were observed in all mice. However, differences of areas of cyst and lesion, thickness of glial scar were significant between hpDCs group and the others.3. IHC found that hypertrophic astrocyte constituting the glial scar around the cyst, were fewer and less hypertrophic in hpDCs group than in DCs or hp groups. Duration of Nestin expression in lesion was 8 weeks in hpDCs group, which were 4 weeks in DCs and hp groups after SCI(p<0.05).4. No axon passed through glial scar or reached cyst in all groups. Conclusion:1. In present study, the model of contused injury in mouse can imitate the clinical changes of SCI.2. Injection of hpDCs can improve functional recovery from SCI in mouse, while pure DCs and hp have less effect.3. Injection of hpDCs into peritoneum has the same effect as into lesion site, showing hpDCs stimulates systemic immunity.4. HpDCs delays the formation, decreases the density and thickness of glial scar, reduces the areas of lesion and cyst, while increases the duration of Nestin expression, and strengthens spontaneous regeneration of neural cells.5. HpDCs can`t make axon passed through glial scar or reached cyst, showing axon regeneration is not the major effect of hpDCs in the model.6. Better outcome of hpDCs group than the others shows insufficient potency of DCs in lesion site, or limit of combination of hp and DCs.