The Role Of Different Immune Status In The Repair Of Spinal Cord Injury In Mice

The role of immunoreactions is against the external environment and maintaining its normal physiological function. When tissue is injured, it not only takes part in resisting infection, but also helping the regeneration and repair of the injured tissue through secreting cytokines. But the immunoreactions have controversial role after spinal cord injury: On the one hand, inflammatory response activated by spinal cord injury(SCI) may worsen the secondary injury; On the other hand, the immune response can clear the cell debris and secret several cytokines such as transforming growth factor-β(TGF-β), brain-derived neurotrophic factor (BDNF), neurotrophin -3 (NT-3) and nerve growth factor (NGF), etc, that can protect neurons from injury and help spinal cord injury repairing and regenerating.It is not suitable to that the autoimmunity inflammation after spinal cord injury is good or bad. Because immune and inflammatory cells can contact mutually and influence the formation of networks, and then exert the role of immune cells. And then formatting those special immune statuses, that plays a distinctive role. Different immune states may have very different immune responses.Our previous experiments showed that the immune inflammatory response may have double faces in spinal cord injury. Early after spinal cord injury, they mainly display their destructive role and worsen spinal cord injury; and at latter time, course of the advance, they gradually transformed into neural protective role and promoting repair of injured tissues. This leads us to wonder whether the injured area has changed its immune state, resulting in the role of immune and inflammatory response transformed into another condition. If the change does exist, and then which types of cells play a key role?To confirm the assumption that we put forward, we tried to analysis the mice's locomotion recovery with different immune status after spinal cord injury, looking for advanced understanding of the various immune status'influence on spinal cord injury repair. And then we can distinguish their different and hunt for the better immune status for spinal cord injury repair and the crucial cells or molecular. We are aim to explore the intervention strategies by tune the immune response for a better treatment after spinal cord injury.In our experiment, we used three kinds of immune status mice. Those are BALB/c, DO11.10 and nude mice. As we known, BALB/c mice have normal immune, while DO11.10 mice and nude mice are transgenic mice of BALB/c genetic background. DO11.10 mice are missing the normal function of peripheral CD4+ T lymphocytes and nude mice are athymic and lack of T lymphocytes. Our experiment was divided into two parts:1. The different functional recovery between the mice with different immune situation after spinal cord injury. We evaluate the recovery of locomotion function behavior of mice with different immune state in mice by the BBB score and footprint experiments through a long time as 11 w after spinal cord injury.2. The cellular and morphological differences between the mice with different immune situation after spinal cord injury.First, at the time points of 1 w, 2 w and 3 w after SCI, we use H & E staining, immunofluorescence staining of macrophages and T lymphocytes in three labeling experiments to analyze the immune cells difference of the spinal cord injury areas in the different immune status mice.And then we used residual nerve fiber 160 (NF160) and glial fibrillary acidic protein (GFAP) immunofluorescence experiments to compare the three kinds of mice'morphological differences of focal spinal cord damage region.We get the following results by the above two parts experiments:1. Three kinds of immune status have significant impact on locomotions recovery after spinal cord injury in mice.First of all, from the BBB scoreing result, we can see the three different mice'recovery of motor function as follow: Nude mice are significantly better than BALB/c and DO11.10 mice (p <0.01); Before 4 weeks after injury, DO11.10 mice are better than the of BALB/c mice (p <0.05), but after 4 weeks BALB / c mice recovered rapidly. What's more at 6 w BALB/c and DO11.10 mice are almost tied and at the 7 w BALB/c mice already much better than DO11. 10 mice(p <0.01).Footprint experiments also confirmed that the three different immune statuses play a important role on spinal cord injury recovery of mice. 2 w after SCI, BALB / c mice'hindlimb position is still very poor, and almost no movement of any jonit; DO11.10 mice were slightly better, and there are obvious trends to move and a certain frequency of the leftside and rightside exchanges; Although Nude mice are less constant, but they are much more better than BALB/c and DO11.10. What's more, the nude mice already have leftside and rightside coordinated motion unsteadily. At the 3 w time point, those three groups of mice have shown their corresponding improvements. BALB/c group has some certain of walking, even though it is mainly produced by the dorsal foot with occasional foot landing; DO11.10 mice appears one side plantar landing and some certain of coordination; Nude mice remains the most robust, with basically using all the foot landing, and its coordination are much better than 2 w's situation. The statistical analysis of the step and step spacing at 6 w and 11 w is basically consistent with the BBB score. Nude mice was obviously better than the BALB/c and DO11.10 group (p <0.01). At 11 w time point, BALB/c mice are slightly better than DO11.10 mice, but no significant difference in statistical analysis.2. The mice with different immune status have significantly different immune cells in the spinal cord injury areas.1 w after injury, H & E staining showed that DO11.10 mice and nude mice have more inflammatory cells than BALB/c mice in the damage zone and approaching area. Macrophages immunofluorescence experiments showed that there are more M2 type macrophages in DO11.10 mice and nude mice damage zone than in BALB/c mice(P<0.01). And nude mice have much more CD11b-positive macrophages / microglia in the damage zone and surrounding than BALB/c and DO11.10 mice(P<0.01). M1 type macrophages are no significant difference in BALB/c and DO11.10 mice. T lymphocytes in the three labeled immunofluorescence experiments confirmed that the mice with different immune status are quite different in the T lymphocyte number. At 3 w time point, BALB / c mice have a large number of activated CD4+, CD8+ T lymphocytes in spinal cord injury area; DO11.10 mice'damage zone exists only individual CD4+ T lymphocytes, but no activity in the state; Nude mice are almost no T lymphocytes appearing in the damage zone and the surrounding.2 w after SCI, NF160 and GFAP immunofluorescence experiments confirmed that those three groups with immune status exist morphological differences in the areas of local spinal cord injury. GFAP immunofluorescence experiments showed that DO11.10 mice and Nude mice damage boundaries clearer. And NF160 positive nerve fibers in the spinal cord injury area of nude mice are much larger.With the above two experiments, we supose that those three different immune statuses of BALB/c, DO11.10 and athymic nude mice have a great impact on the locomotion recovery from their spinal cord injury. The immune status of nude mice is more conducive to their recovery from spinal cord injury. We found that the high level of arginaseⅠ, CD11b positive macrophages and T lymphocytes expression in immune deficiency state may be more beneficial for the repair of injuried spinal cord through analysis of three kinds of immune immune cells in the spinal cord injury area. We conclude that the complex function of T lymphocyte and the particular immune status is very relevant with the special recovery from spinal cord injury of BALB/c mice.