Fractalkine Receptor Deficiency Is Associated with Early Protection but Late Worsening of Outcome following Brain Trauma in Mice - PubMed (original) (raw)

Fractalkine Receptor Deficiency Is Associated with Early Protection but Late Worsening of Outcome following Brain Trauma in Mice

Elisa R Zanier et al. J Neurotrauma. 2016.

Abstract

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.

Keywords: fractalkine receptor; inflammation; macrophages; microglia; traumatic brain injury.

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Figures

<b>FIG. 1.</b>

**FIG. 1.

Experimental design. (A) Pattern of microglia/macrophage (M/μ) activation post–traumatic brain injury (TBI) in wild type (WT) mice. Mice were sacrificed 1 day, 2 days, 4 days, 7 days, or 5 weeks after TBI/sham injury for immunohistochemical analysis. (B) Consequences of fractalkine receptor deletion (CX3CR1−/−) on behavioral and histopathological outcome, and on M/μ activation post-TBI. Sensorimotor deficits evaluation and histological analysis were performed in WT and CX3CR1−/− mice 4 days and 5 weeks after TBI/sham injury. Mice were sacrificed at 4 days, 7 days, or 5 weeks for M/μ immunohistochemistry and morphological analysis or at 1 day, 4 days, or 5 weeks for CD11b+ cell sorting by MACS technology and gene expression analysis. Color image is available online at

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<b>FIG. 2.</b>

**FIG. 2.

Quantitative immunohistochemical analysis of microglia/macrophage (M/μ) markers in wild type (WT) mice after traumatic brain injury (TBI)/sham injury. Quantification of CD11b (A), CD68 (B), inducible nitric oxide synthase (iNOS) (C), and Ym1 (D) immunostaining in WT mice 1 day, 2 days, 4 days, 7 days, and 5 weeks after TBI/sham injury. Above, representative microphotographs of each marker in sham and TBI mice at the time-point of marker maximal expression and at 5 weeks (bar = 20 μm). Below, quantification of the staining. Data are expressed as mean ± standard deviation of 27 frames/mouse, n = 8. *p < 0.05 vs sham; ^^p < 0.01, ^^^p < 0.001 vs. 4 days for all selected markers, one-way analysis of variance followed by Tukey's post hoc test. n.d., not detected.

<b>FIG. 3.</b>

**FIG. 3.

Early and late outcome in wild type (WT) and fractalkine receptor deletion (CX3CR1−/−) mice post–traumatic brain injury (TBI). (A) Functional damage assessed by neuroscore. Before TBI, CX3CR1−/− and WT mice showed a similar neuroscore. After TBI, CX3CR1−/− mice showed a significant reduction of sensorimotor deficits at an early time-point (4 days post-TBI), compared with WT mice. Recovery over time was detected in WT but not in CX3CR1−/− mice, thus leading to a significantly worst performance of CX3CR1−/− mice, compared with WT mice at 5 weeks. (B) Contusion volume. Over time, both WT and CX3CR1−/− mice showed progressive cortical tissue loss with no difference between genotypes. (C, D) DNA damage. WT but not CX3CR1−/− mice showed a decrease in the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)+ cells over time post-TBI. Compared with WT mice, CX3CR1−/− mice showed a significant decrease in TUNEL+ cells at 4 days (C) as shown in the representative microphotographs (D). (E, F) Neuronal count. CX3CR1−/− but not WT mice showed a cell loss over time in traumatized cortex. At 5 weeks post-TBI, CX3CR1−/− mice showed a significant lower number of neuronal cells, compared with WT mice (E), as shown in the representative microphotographs (F). Fields for TUNEL evaluation and quantification and for neuronal counts were positioned as depicted in the D and F, respectively. Data are expressed as mean ± standard deviation, n = 11. *p < 0.05, ###p < 0.001, two-way analysis of variance followed by Tukey's post hoc test. Color image is available online at

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<b>FIG. 4.</b>

**FIG. 4.

Morphological analysis of microglia/macrophage (M/μ) in wild type (WT) and fractalkine receptor deletion (CX3CR1−/−) mice 4 days and 5 weeks after traumatic brain injury (TBI)/sham injury. CD11b+ cells showed an increase of the area (A) and the perimeter (B) at 4 days after TBI in WT mice, followed by a decrease at 5 weeks post-injury. Compared with WT mice, CX3CR1−/− mice showed a significant reduction of CD11b+ cell area (A) and perimeter (B) at 4 days followed by a significant increase of both parameters 5 weeks after TBI. Representative microphotographs show the typical morphology of M/μ cells in WT and CX3CR1−/− mice at 4 days (C) and 5 weeks (D). Data are reported as mean ± SD, n = 8. **p < 0.01, ***p < 0.001. Two-way analysis of variance followed by Tukey's post hoc test.

<b>FIG. 5.</b>

**FIG. 5.

Quantitative immunohistochemical analysis of microglia/macrophage (M/μ) markers in wild type (WT) and fractalkine receptor deletion (CX3CR1−/−) mice 4 days and 5 weeks after traumatic brain injury (TBI)/sham injury. (A) No difference between genotypes was detected for CD11b+ immunostaining. (B) Compared with WT mice, CX3CR1−/− mice showed a reduction of lysosomal activity (CD68+ positivity) at 4 days post-TBI. (C) Compared with WT mice, CX3CR1−/− mice showed a significant decrease (4 days) followed by a significant increase (5 weeks) in the density of inducible nitric oxide synthase (iNOS)+ cells. (D, E-a). No differences between genotypes were detected for Ym1 and high expression of CD45 (CD45high) immunostaining. (E-b) Representative microphotograph showing the typical appearance of round-shaped CD45high cells (white arrow) selectively counted. Data are reported as mean ± standard deviation (n = 8). *p < 0.05, ***p < 0.001, two-way analysis of variance followed by Tukey's post hoc test. n.d., not detected.

<b>FIG. 6.</b>

**FIG. 6.

Coexpression of CD11b (red) and CD68 (green) 4 days post–traumatic brain injury in wild type (WT) and fractalkine receptor deletion (CX3CR1−/−) mice. CD68 was greatly increased in CD11b+ cells displaying a hypertrophic morphology in WT mice. CD68 often co-localized with the membrane marker CD11b in WT mice, while it remained mainly located in the cytoplasm in CX3CR1−/− mice. Data are representative of three independent experiments. Bars: 20 μm. Color image is available online at

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<b>FIG. 7.</b>

**FIG. 7.

Gene expression analysis of CD11b+ sorted cells in wild type (WT) and fractalkine receptor deletion (CX3CR1−/−) non-injured (sham) mice. (A, B) Compared with WT sham, CX3CR1−/− sham mice had higher interleukin (IL) 1β and tumor necrosis factor (TNF) α messenger RNA expression. (C, D) No differences between genotypes were detected for sham expression of insulin-like growth factor 1 (IGF1) and IL10. Data are expressed as fold of induction, compared with the WT sham group, and are reported as mean ± standard deviation (n = 8). ***p < 0.001, Mann Whitney _t_-test.

<b>FIG. 8.</b>

**FIG. 8.

Gene expression analysis of CD11b+ sorted cells in wild type (WT) and fractalkine receptor deletion (CX3CR1−/−) mice 1 day, 4 days, and 5 weeks after traumatic brain injury (TBI). Compared with WT mice, CX3CR1−/− mice displayed significantly higher insulin-like growth factor 1 (IGF1) and interleukin (IL) 10 messenger ribonucleic acid (mRNA) expression 1 day post-TBI (C, D). At 4 days and 5 weeks, however, a switch in IGF1 mRNA expression was observed, being IGF1 significantly lower in CX3CR1−/− mice. Data are expressed as fold of induction, compared with the WT sham group, and are reported as mean ± standard deviation (n = 8). *p < 0.05 vs. WT, two-way analysis of variance followed by Tukey's post hoc test.

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