Chemokine (C-C motif) receptor 5 (CCR5) is expressed not only in the immune cells but also in cerebral cells such as neurons, glia, and vascular cells. Stroke triggers high expression of CCR5 in the brain. However, the role of CCR5 in stroke remains unclear. In this study, using bone marrow chimeras we have determined the involvement of brain-derived or bone marrow-derived CCR5 in neuroprotection and brain repair after experimental stroke. CCR5-/- mice that received either wild-type (WT) or CCR5-/- bone marrow transplantation showed larger infarction sizes than the WT mice that received either WT or CCR5-/- bone marrow transplantation in both the acute (48h) and subacute (2 months) phases after cerebral cortical ischemia, suggesting that the lack of CCR5 in the brain leads to severe brain damage after stroke. However, the lack of CCR5 in the bone marrow-derived cells did not affect infarction size. The impairments of somatosensory-motor function and motor coordination were exacerbated in the mice lacking CCR5 in the brain. At 2 months post-stroke, increased degenerative neurons, decreased dendrites and synapses, decreased Iba1+ microglia/ macrophages, reduced myelination and CNPase+ oligodendrocytes in the peri-infarct cortex were observed in the mice lacking CCR5 in the brain. These pathological changes are significantly correlated with the increased infarction size and exacerbated neurological deficits. These data suggest that brain-derived CCR5 plays a key role in neuroprotection and brain repair in the subacute phase of stroke. This study reveals a novel role of CCR5 in stroke, which sheds new light on post-stroke pathomechanism.
Figure 1. Experimental flowchart and groups. (A) A schematic diagram shows the flowchart of the experiment. Three-month-old wild type (WT) and CCR5-/- mice received irradiation and bone marrow transplantation. After one month-bone marrow reconstruction, mice were subjected to middle cerebral artery occlusion (MCAO) for induction of focal cerebral ischemia. Triphenyl tetrazolium chloride (TTC) staining was performed 48h after MCAO to measure the infarction size (N = 6/group). Two weeks after MCAO, behavioral tests were performed to evaluate neurological deficits (N=9/group). Two months after MCAO, mice were sacrificed for immunohistochemistry (N=7-8/group). (B) A table shows the information about the four experimental groups of chimeric mice. RwtDwt: WT mice received bone marrow transplantation (BMT) from the bone marrow donor of WT mice. RwtDccr5: WT mice received BMT from the bone marrow donor of CCR5-/- mice. Rccr5Dwt: CCR5-/- mice received BMT from the bone marrow donor of WT mice. Rccr5Dccr5: CCR5-/- mice received BMT from the bone marrow donor of CCR5-/- mice.
Figure 2. Infarct volume is increased in the mice lacking CCR5 in the brain. (A) Representative images of triphenyl tetrazolium chloride (TTC) staining 48h after middle cerebral artery occlusion (MCAO). (B) Quantification data of infarct volume using TTC staining. (C) Representative images of cresyl violet staining two months after MCAO. A longitudinal notch was made in the left brain to distinguish the contralesional hemisphere from the ipsilesional hemisphere. (D) Quantification data of infarct volume using cresyl violet staining. Mean ± S.E.M. N=6/group. **p<0.01, ***p<0.001. One-way ANOVA with Tukey post hoc test.
Figure 3. Cerebral cortical ischemia-induced neurological deficits are exacerbated in the mice lacking CCR5 in the brain. (A) Somatosensory-motor function was examined using a tape removal test. Data were collected from three independent trials each day. Mean ± S.E.M. N=9/group. *p<0.05, **p<0.01. One-way ANOVA with Tukey post hoc test. (B) Motor coordination function was examined using a rotarod test. Data were collected from three independent trials each day for five consecutive days. Mean± S.E.M. N=9/group. *p<0.05, **p<0.01; RwtDwt mice vs. Rccr5Dwt mice. &p<0.05, RwtDwt mice vs. Rccr5Dccr5 mice. #p<0.05, RwtDccr5 mice vs. Rccr5Dwt mice. Two-way repeated ANOVA with Fisher’s LSD multiple comparison test.
Figure 4. Neuronal degeneration in the peri-infarct cortex 2 months after cerebral cortical ischemia. Note that the mice lacking CCR5 in the brain show increased Fluoro-Jade C+ degenerating neurons, reduced MAP2+ dendritic density and decreased PSD-95+ post-synapses in the peri-infarct cortex. (A-D) Representative images show immunofluorescence staining for NeuN in the peri-infarct cortex. (E) Quantification data of NeuN positive neurons in the peri-infarct cortex. (F-I) Representative images show Fluoro-Jade C staining in the peri-infarct cortex. (J) Quantification data of Fluoro-Jade C positive degenerating neurons in the peri-infarct cortex. (K-R) Representative images show MAP2+ dendrites and PSD-95+ post-synapses in the peri-infarct cortical layer 1-3. (S) Schematic diagrams indicate the imaging areas in the cortex. (T-W) Quantification data of MAP2 and PSD-95 positive area in the peri-infarct cortical layer 1 and layer 2/3. Mean± S.E.M. N=7-8/group. *p<0.05, **p<0.01. One-way ANOVA with Tukey post hoc test. N.S.: not significant.
Figure 5. Inflammatory cells in the peri-infarct cortex 2 months after cerebral cortical ischemia. Note that reduced Iba1+ cells, increased P2RY12+ cells and decreased CD68+ cells in the peri-infarct cortex are seen in the mice lacking CCR5 in the brain (Rccr5Dwt). (A-E) Representative images of immunofluorescence staining for Iba1 in the peri-infarct cortex. (F-J) Representative images of immunofluorescence staining for P2RY12 in the peri-infarct cortex. (K-O) Representative images of immunofluorescence staining for CD68 in the peri-infarct cortex. (P) Schematic diagrams show the imaging areas in the cortex. (Q-S) Quantification data of Iba1, P2RY12 and CD68 positive area in the peri-infarct cortex. Mean± S.E.M. N=7-8/group. *p<0.05, **p<0.01, *** p<0.001. One-way ANOVA with Tukey post hoc test.
Figure 6. Neuroinflammatory molecule expression in the peri-infarct cortex 2 months after cerebral cortical ischemia. Note that stroke mice show reduced IL-4 expression and increased NOS2 expression in the peri-infarct cortex as compared to the naïve control mice. Stroke mice lacking CCR5 in both the brain and bone marrow show increased NOS2 expression in the peri-infarct cortex (RwtDccr5, Rccr5Dwt, and Rccr5Dccr5 vs. RwtDwt). (A-E) Representative images of immunofluorescence staining for IL-4 in the peri-infarct cortex. (F to J) Representative images of immunofluorescence staining for NOS2 in the peri-infarct cortex. (K) Schematic diagrams indicate the imaging areas in the cortex. (L and M) Quantification data of IL-4 and NOS2 positive area in the peri-infarct cortex. Mean± S.E.M. N=7-8/group. *p<0.05, **p<0.01. One-way ANOVA with Tukey post hoc test.
Figure 7. Myelination in the peri-infarct cortex 2 months after cerebral cortical ischemia. Note that mice lacking CCR5 in the brain show reduced myelination in the peri-infarct cortex 2 months after cerebral cortical ischemia. (A-D) Representative images of immunofluorescence staining for CNPase in the peri-infarct cortex. (E-H) Representative images of immunofluorescence staining for MBP in the peri-infarct cortex. (I) A schematic diagram indicates the imaging areas in the cortex. (J and K) Quantification data of CNPase and MBP positive areas in the peri-infarct cortex. Mean± S.E.M. N=7-8/group. *p<0.05, **p<0.01. One-way ANOVA with Tukey post hoc test.
Figure 8. Blood vessel density and astrogliosis in the peri-infarct cortex are not affected by CCR5 deficiency. (A-D) Representative images show Lectin positive blood vessels in the peri-infarct cortex. (E-H) Representative images of immunofluorescence staining for GFAP in the peri-infarct cortex. (I) Schematic diagrams indicate the imaging areas in the cortex. (J and K) Quantification data of Lectin and GFAP positive area in the peri-infarct cortex. Mean ± S.E.M. N=7-8/group. One-way ANOVA with Tukey post hoc test. N.S.: not significant.
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