Resumen de Experimental spinal cord injury: modulation of inflammatory response and its effects on secondary damage
Injury to the spinal cord results in an immediate primary damage followed by a secondary phase of tissue damage that occurs over a period of several weeks, which contributes significantly to permanent functional disabilities. The mechanisms of the secondary injury are multiple and not fully defined; however, a large number of studies suggest that the inflammatory response occurring after spinal cord injury (SCI), is one of the main contributors to secondary damage. Responses to injury, however, are also accompanied by protective mechanisms that antagonize the damaging events or mediate repair. The final outcome of the SCI will therefore depend on the balance between the harmful and helpful responses. Therefore potentiating protective mechanisms can be expected to substantially reduce functional disability after SCI. Although regeneration of disrupted axons and replacement of damaged neurons and oligodendrocytes after SCI are important goals to promote functional recovery, prevention of the secondary damage to axons, neuronal cell bodies, myelin and glial cells that follows the initial trauma is likely to be more easily amenable to treatment.
Work 1: Nerve Injury-Induced protein (Ninjurin-1) is an adhesion molecule found to be up regulated in DRG neurons and Schwann cells (SCs) after nerve injury to promote neurite outgrowth. We established collaboration with Dr Alexandre Prat at Université de Montreal and performed a proteomic analysis of lipid raft membrane microdomains isolated from human brain endothelial cells and identified Ninjurin-1 as an important candidate for leukocyte transmigration into the CNS. We demonstrated up-regulation of Ninjurin-1 on BBB-ECs, infiltrating macrophages and CD11c+ dendritic cells during the course of experimental allergic encephalomyelitis (EAE), and in active multiple sclerosis lesions. In addition, Ninjurin-1 blocking peptide (Ninj26-37) significantly restricts the migration of human CD14+ monocytes across BBB-ECs, without affecting lymphocyte recruitment. We show that daily injections of Ninj26-37 during the course of EAE reduce infiltration of macrophages and dendritic cells and decrease the clinical and histopathological severity of EAE. This study therefore demonstrates that Ninjurin-1 plays a specific role in the recruitment of myeloid antigen-presenting cells during neuroinflammation and identifies its potential use as a target to limit CNS inflammation. Our data demonstrate that Ninjurin-1 is up-regulated after SCI, peaking at day 7. Similar to EAE, Ninjurin-1 is also expressed in endothelial cells, macrophages/microglia and dendritic cells, but also in oligodendrocytes and neurons in the contused spinal cord. Treatment with Ninj-26-37 reduced the accumulation of microglia/macrophages in the spinal cord parenchyma at day 7 post-injury. In contrast to EAE, we show that the reduction in myeloid cell recruitment does not lead to functional recovery and tissue protection in SCI.
Work 2: A recent work has revealed that alphaB-crystallin (CRYAB), a small heat shock protein, ameliorates EAE and brain ischemia by acting as a negative regulator of adaptive immune response. However, new evidences also revealed that CRYAB triggers the activation of innate immune response in multiple sclerosis. Interestingly, upon CRYAB stimulation, microglial cells adopt an anti-inflammatory phenotype, suggesting that they could mediate repair rather than harmful effects. We assessed whether administration of rhCRYAB enhanced functional recovery and tissue sparing in SCI. We observed that treatment with rhCRYAB led to neuroprotection and to improved motor function when given after SCI. Interestingly, the beneficial effects of CRYAB in SCI were accompanied with an up regulation in the expression of several cytokines and chemokines at 12 hours after lesion, greater recruitment of neutrophils in the spinal cord parenchyma, and reduced influx of ¿inflammatory¿ monocytes. Although the role of ¿inflammatory¿ monocytes in SCI is not known yet, a recent work demonstrated that they contribute to the course of EAE, and thus, they may exert a similar harmful effect in SCI.
Work 3: Contrary to activated macrophages in most CNS disorders, activation of macrophages triggered when LPC is injected into the spinal cord does not cause axonal damage, suggesting that LPC may induce a non-cytotoxic activation of macrophages. In collaboration with Samuel David¿s laboratory at McGill Univeristy, we are studying whether macrophages isolated from spinal cords after injury or LPC injection are phenotypically different, and whether LPC could drive macrophages activation towards a non-cytotoxic phenotype when injected into the injured spinal cord. We revealed by doing an Affimetrix GeneArray that macrophages isolated from the spinal cord at the peak of inflammation after SCI or LPC injection are different. Although deeper analysis on the data obtained from this GeneArray is needed, we have identified some specific genes that are up regulated in macrophages isolated from the contused spinal cord which could be potential markers of ¿cytotoxic¿ macrophages (KCNN4, ATPase d2, CCL14, CD11c and syntatin-6). All these genes have shown to exert toxicity in several models of CNS disorders, but their role in SCI remains unknown. However, recent data from Samuel David¿s laboratory revealed that pharmacological inhibition of KCNN4 by TRAM-34 after contusion SCI led to greater functional and histological outcomes. In addition, the role of ATPase d2 and mfge8 is currently being studied in Samuel David¿s laboratory. Whether LPC may modify the activation of macrophages after SCI and down-regulate these cytotoxic genes is not known yet. We showed that intraspinal injection of LPC into the contused spinal cord promoted functional recovery and tissue protection, and altered the profile of cytokine/chemokine expression but not the recruitment of myeloid cells after SCI. These data suggest that LPC may drive activation of macrophages towards a non-cytotoxic phenotype when injected into the injured spinal cord. However, further experiments assessing whether macrophages isolated from contused spinal cord injected LPC have reduced the expression of the potential cytotoxic markers highlighted above, among others, have to be carried out in the laboratory.
In future experiments we will extract mRNA from macrophages purified from contused spinal cords that received either LPC or saline injection. We will assess whether intraspinal injection of LPC modulates gene expression profile in macrophages after SCI, and will drive inflammation from a cytotoxic towards a non-cytotoxic phenotype, as observed after LPC injection in the naïve spinal cord.
