Targeting the pro-inflammatory and cytotoxic environment to improve functional recovery after Spinal Cord Injury

Abstract

Spinal cord injury and the resulting paralysis are critical unsolved problems worldwide. The primary injury develops into a secondary injury cascade for which currently no treatment can help. The secondary injury is characterized by an inflammatory response, leading to the infiltration and activation of immune cells. Later on, the formation of a glial scar is a major bottleneck for regenerative processes. To enhance functional recovery, immunomodulation must be accompanied by regenerative processes like neurite outgrowth, angiogenesis and degradation of the scar tissue, amongst others. It is clear that a multifactorial approach is needed, but first in depth investigation of different mechanisms that can increase functional regeneration is necessary. As a first approach, we modulated the stress systems after SCI. As described in chapter 2, inevitably the sympathetic nervous system (SNS) of the stress systems will be activated after SCI. This will cause hormonal and metabolic changes with direct and indirect effects on the inflammatory system. Next, the inflammatory response will play a major part in the increase of the secondary injury and the loss of function. In chapter 3 we hypothesized that stimulating the β-adrenergic pathway (part of the SNS) provides neuroprotection and neuronal outgrowth in order to stimulate functional regeneration after SCI. However, β-AR agonism with clenbuterol (β2- AR agonist) or xamoterol (β1-AR agonist) did not affect functional recovery or neuronal viability and outgrowth. Summarized, the detrimental effects of β-AR antagonism with propranolol on primary neuron viability in vitro and functional recovery and helper T cells in vivo let us suggest the β-adrenergic pathway is indispensable for SCI recovery. Secondly, we aimed to improve functional revascularization after SCI via modulation of the β-adrenergic pathway. We show that terbutaline promotes angiogenesis by stimulating the β2-AR on bEnd.3 cells in vitro and stimulates blood vessel formation in the CAM assay in ovo, with only a suggestive, nonsignificant improvement of the functional outcome in vivo (chapter 4). Taken together, β-AR stimulation did not improve functional recovery after SCI, although it stimulated blood vessel formation ex vivo and inhibition showed detrimental effects on neuron viability. Therefore, we conclude that β-adrenoceptor modulation is not an effective therapeutic strategy to improve functional outcome after SCI. In a third approach, we modulated the inflammatory response directly. In chapter 5, we show that perilesional transfer of IL-13 secreting macrophages (IL-13-Mɸ) is a successful experimental treatment to improve functional and histopathological recovery in the SCI mouse model. We suggest that the injected anti-inflammatory IL-13-Mɸs migrate to the lesion site and convert the local destructive M1-Mɸs to M2, creating a neuroprotective environment more prone to functional regeneration. We showed an increase in the number of neuroprotective arg-1 positive microglia/macrophages and a decrease in the number of axon-attacking MHCII positive microglia/macrophages by IL-13-Mɸ treatment at the lesion site. In addition, there was a decrease in the number of macrophage-axon contacts in the IL-13-Mɸ treated mice, suggesting a reduction in axonal dieback. Hence, we speculate that a reduction in axonal dieback may have led to the improved histopathological and functional outcome. Lastly, SCI is characterized by the formation of a glial and fibrotic scar at the lesion site. This scar creates a major barrier for regenerating axons and contributes significantly to the impaired functional outcome. Therefore, we have investigated the effects of mMCP4 on scarring and recovery after SCI by using mMCP4 knockout mice. The results described in chapter 6 suggest that mast cells promote scar remodeling after SCI via mMCP4. However, it remains speculative whether mMCP4 cleaves ECM components directly or indirectly after injury. It might be that the immunomodulatory effects of mMCP4 may indirectly suppress the scarring response after CNS injury. For example, the absence of mMCP4 increased the expression of pro-inflammatory cytokines (e.g. IL-6) which, in turn, can increase the deposition of CSPGs. In summary, our data imply a new potential scar-remodeling mechanism via which mMCP4 can support recovery after CNS injury, in addition to the immunomodulary effects that we have reported before. Future research should aim at defining the therapeutic validity of MCPs (e.g. application of recombinant MCP4) to improve recovery after SCI. In conclusion, we have demonstrated in this thesis that immunomodulatory therapy using IL-13 secreting macrophages provides a great therapeutic potential for the treatment of SCI. However, further research is still required to identify the mechanisms behind these effects. SCI needs a multifactorial approach, therefore we have focused on different strategies to tackle the damage to different mechanisms caused by the secondary injury. The focus of future research should be combination treatments, starting with the ones that have singular positive effects and are unlikely to interact with each other.