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An impaired ability to regulate microglia activation by fractalkine (CX3CL1) leads to microglia chronic sub-activation. How this condition affects outcome after acute brain injury is still debated, with studies showing contrasting results depending on the timing and the brain pathology. Here, we investigated the early and delayed consequences of fractalkine receptor (CX3CR1) deletion on neurological outcome and on the phenotypical features of the myeloid cells present in the lesions of mice with traumatic brain injury (TBI). Wild type (WT) and CX3CR1(-/-) C57Bl/6 mice were subjected to sham or controlled cortical impact brain injury. Outcome was assessed at 4 days and 5 weeks after TBI by neuroscore, neuronal count, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Compared with WT mice, CX3CR1(-/-) TBI mice showed a significant reduction of sensorimotor deficits and lower cellular damage in the injured cortex 4 days post-TBI. Conversely, at 5 weeks, they showed a worsening of sensorimotor deficits and pericontusional cell death. Microglia (M) and macrophage (μ) activation and polarization were assessed by quantitative immunohistochemistry for CD11b, CD68, Ym1, and inducible nitric oxide synthase (iNOS)-markers of M/μ activation, phagocytosis, M2, and M1 phenotypes, respectively. Morphological analysis revealed a decreased area and perimeter of CD11b(+) cells in CX3CR1(-/-) mice at 4 days post-TBI, whereas, at 5 weeks, both parameters were significantly higher, compared with WT mice. At 4 days, CX3CR1(-/-) mice showed significantly decreased CD68 and iNOS immunoreactivity, while at 5 weeks post-injury, they showed a selective increase of iNOS. Gene expression on CD11b(+) sorted cells revealed an increase of interleukin 10 and insulin-like growth factor 1 (IGF1) at 1 day and a decrease of IGF1 4 days and 5 weeks post-TBI in CX3CR1(-/-), compared with WT mice. These data show an early protection followed by a chronic exacerbation of TBI outcome in the absence of CX3CR1. Thus, longitudinal effects of myeloid cell manipulation at different stages of pathology should be investigated to understand how and when their modulation may offer therapeutic chances.

Many efforts have been performed in order to understand the role of recruited macrophages in the progression of spinal cord injury (SCI). Different studies revealed a pleiotropic effect played by these cells associated to distinct phenotypes (M1 and M2), showing a predictable spatial and temporal distribution in the injured site after SCI. Differently, the role of activated microglia in injury progression has been poorly investigated, mainly because of the challenges to target and selectively modulate them in situ. A delivery nanovector tool (poly-ε-caprolactone-based nanoparticles) able to selectively treat/target microglia has been developed and used here to clarify the temporal and spatial involvement of the pro-inflammatory response associated to microglial cells in SCI. We show that a treatment with nanoparticles loaded with minocycline, the latter a well-known anti-inflammatory drug, when administered acutely in a SCI mouse model is able to efficiently modulate the resident microglial cells reducing the pro-inflammatory response, maintaining a pro-regenerative milieu and ameliorating the behavioral outcome up to 63 days post injury. Furthermore, by using this selective delivery tool we demonstrate a mechanistic link between early microglia activation and M1 macrophages recruitment to the injured site via CCL2 chemokine, revealing a detrimental contribution of pro-inflammatory macrophages to injury progression after SCI., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Microglia/macrophages (M) are major contributors to postinjury inflammation, but they may also promote brain repair in response to specific environmental signals that drive classic (M1) or alternative (M2) polarization. We investigated the activation and functional changes of M in mice with traumatic brain injuries and receiving intracerebroventricular human bone marrow mesenchymal stromal cells (MSCs) or saline infusion. MSCs upregulated Ym1 and Arginase-1 mRNA (p < 0.001), two M2 markers of protective M polarization, at 3 and 7 d postinjury, and increased the number of Ym1(+) cells at 7 d postinjury (p < 0.05). MSCs reduced the presence of the lysosomal activity marker CD68 on the membrane surface of CD11b-positive M (p < 0.05), indicating reduced phagocytosis. MSC-mediated induction of the M2 phenotype in M was associated with early and persistent recovery of neurological functions evaluated up to 35 days postinjury (p < 0.01) and reparative changes of the lesioned microenvironment. In vitro, MSCs directly counteracted the proinflammatory response of primary murine microglia stimulated by tumor necrosis factor-α + interleukin 17 or by tumor necrosis factor-α + interferon-γ and induced M2 proregenerative traits, as indicated by the downregulation of inducible nitric oxide synthase and upregulation of Ym1 and CD206 mRNA (p < 0.01). In conclusion, we found evidence that MSCs can drive the M transcriptional environment and induce the acquisition of an early, persistent M2-beneficial phenotype both in vivo and in vitro. Increased Ym1 expression together with reduced in vivo phagocytosis suggests M selection by MSCs towards the M2a subpopulation, which is involved in growth stimulation and tissue repair.