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Aging and disease
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Age-related CCL12 Aggravates Intracerebral Hemorrhage-induced Brain Injury via Recruitment of Macrophages and T Lymphocytes
Jiacheng Huang1, Guoqiang Yang1, Xiaoyi Xiong1, Maolin Wang2, Junjie Yuan1, Qing Zhang1, Changxiong Gong1, Zhongming Qiu1, Zhaoyou Meng, Rui Xu1, Qiong Chen1, Ru Chen1, Lexing Xie1, Qi Xie1, Wenjie Zi1, Guohui Jiang1, Yu Zhou1,*, Qingwu Yang1,*
1Department of Neurology, Xinqiao Hospital, the Army Medical University (Third Military Medical University), Chongqing 400037, China.
2Central Laboratory, Xinqiao Hospital, the Army Medical University (Third Military Medical University), Chongqing 400037, China.
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Abstract  

Circulating factors associated with aging have been shown to be involved in the development of age-related chronic and acute brain diseases. Here, we aimed to investigate the roles and mechanisms of CCL12, a circulating factor that is highly expressed in the plasma of elderly patients after intracerebral hemorrhage (ICH) using parabiosis and ICH models. Neurological deficit score (NDS), mortality rate, brain water content (BWC), and levels of inflammatory factors were determined to assess the degree of brain injury-induced damage. Peripheral inflammatory cell infiltration was examined using immunofluorescence and flow cytometry. After confirming that acute brain injury after ICH was aggravated with age, we found that brain and plasma CCL12 levels were markedly higher in old mice than in young mice after ICH, and that plasma CCL12 was able to enter the brain. Using CCL12-/- mice, we showed that the degree of damage in the brain—as determined by NDS, mortality rate, BWC, levels of inflammatory factors, and numbers of degenerative and apoptotic neural cells and surviving neurons was significantly attenuated compared to that observed in old wild-type (WT) mice. These effects were reversed in CCL12-treated old mice. The detrimental effects caused by CCL12 may involve its ability to recruit macrophages and T cells. Finally, the administration of an anti-CCL12 antibody markedly improved the outcomes of ICH mice. Our results are the first to indicate that elevated peripheral CCL12 levels in old mice aggravates ICH-induced brain injury by recruiting macrophages and T cells. Thus, CCL12 may be a new target for ICH treatment.

Keywords age      CCL12      intracerebral hemorrhage      inflammation      parabiosis     
Corresponding Authors: Yu Zhou,Qingwu Yang   
Just Accepted Date: 07 January 2020  
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Jiacheng Huang
Guoqiang Yang
Xiaoyi Xiong
Maolin Wang
Junjie Yuan
Qing Zhang
Changxiong Gong
Zhongming Qiu
Zhaoyou Meng
Rui Xu
Qiong Chen
Ru Chen
Lexing Xie
Qi Xie
Wenjie Zi
Guohui Jiang
Yu Zhou
Qingwu Yang
Cite this article:   
Jiacheng Huang,Guoqiang Yang,Xiaoyi Xiong, et al. Age-related CCL12 Aggravates Intracerebral Hemorrhage-induced Brain Injury via Recruitment of Macrophages and T Lymphocytes[J]. Aging and disease, 10.14336/AD.2019.1229
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http://www.aginganddisease.org/EN/10.14336/AD.2019.1229     OR     http://www.aginganddisease.org/EN/Y/V/I/0
GeneForward primerReverse primer
CCL125'-ATTTCCACACTT
CTATGCCTCCT-3'
5'-ATCCAGTATGGT
CCTGAAGATCA-3'
GAPDH5'-GGTTGTCTC
CTGCGACTTCA-3'
5'-TGGTCCAGGGT
TTCTTACTCC-3'
Table 1  Primers used for quantitative real-time PCR.
Figure 1.  Peripheral CCL12 can enter the brain after ICH. (A) A schematic showing the parabiotic pairings. (B) Peripheral CCL12 protein was detected by ELISA in isochronic WT, heterochronic WT and CCL12-/-, and isochronic CCL12-/- mice. (C) CCL12 mRNA expression was detected in the brain of isochronic (WT-WT and CCL12-/--CCL12-/-) and heterochronic (WT-CCL12-/-) mice after ICH by real-time PCR. (D) CCL12 protein expression was detected by western blot in the perihematomal brain tissues from isochronic WT and heterochronic mice after ICH. The bar graphs show the means ± SDs. n = 5. P-values were determined by ANOVA. *P < 0.05; **P < 0.01; ns, not significant.
Figure 2.  CCL12 knockout increases inflammatory injury in perihematomal brain tissues after ICH. (A-C) NDS (n = 5), mortality rate (B) (n = 20) and BWC (C) (n = 5) in WT and CCL12-/- mice after ICH. (D) IL-6, IL-10 and TNF-αprotein levels in WT and CCL12-/- mice detected by ELISA after ICH (n = 6). The bar graphs show the means ± SDs. P-values were determined by ANOVA. *P < 0.05; **P < 0.01; ns, not significant.
Figure 3.  CCL12 supplementation aggravates ICH-induced brain injury in mice. (A-C) NDS (n = 5), mortality rate (B) (n = 20) and BWC (n = 5) levels of WT, old CCL12-treated and vehicle-treated mice after ICH. (D) IL-6, IL-10 and TNF-α protein levels in WT, old CCL12-treated and vehicle-treated mice were detected by ELISA after ICH (n = 6). The bar graphs show the means ± SDs. n = 5. P-values were determined by ANOVA. *P < 0.05; **P < 0.01; ns, not significant.
Figure 4.  CCL12 knockout increases the number of surviving neurons. (A) H&E staining of perihematomal brain tissues from old WT and CCL12-/- mice after ICH surgery. Scale bars, 200 μm top images and 100 μm bottom images. (B) TUNEL assay results and quantification of apoptotic cells in perihematomal brain tissues derived from WT and CCL12-/- mice after ICH. Scale bars, 100 μm. (C) FJB staining and quantification of degenerative cells in perihematomal brain tissues derived from WT and CCL12-/- mice after ICH surgery. (D) Nissl staining and quantification of Nissl bodies in perihematomal brain tissues derived from WT and CCL12-/- mice after ICH. Scale bars, 200 μm on the top and 100 μm on the bottom. Scale bars, 200 μm top images and 100 μm bottom images. The bar graphs show the means ± SDs. n = 5. P-values were determined by ANOVA. *P < 0.05; **P < 0.01; ns, not significant.
Figure 5.  CCL12 treatment decreases the number of surviving neurons. (A) H&E staining of perihematomal brain tissues from old WT, vehicle-treated and CCL12-treated mice after ICH surgery. Scale bars, 200 μm top images and 100 μm bottom images. (B) TUNEL assay results and quantification of apoptotic cells in perihematomal brain tissues derived from old WT, vehicle-treated and CCL12-treated mice after ICH surgery. Scale bars, 100 μm. (C) FJB staining and quantification of degenerative cells in perihematomal brain tissues derived from old WT, vehicle-treated and CCL12-treated mice after ICH surgery. (D) Nissl staining and quantification of Nissl bodies in perihematomal brain tissues derived from old WT, vehicle-treated and CCL12-treated mice after ICH surgery. Scale bars, 200 μm top images and 100 μm bottom images. The bar graphs show the means ± SDs. n = 5. P-values were determined by ANOVA. *P < 0.05; **P < 0.01; ns, not significant.
Figure 6.  Peripheral CCL12 aggravates the ICH-induced damage by recruiting peripheral macrophages and CD3+ T lymphocytes. (A) Representative fluorescence microscopy images show infiltrating F4/80+ cells (macrophages) in the perihematomal tissues (the arrows indicate F4/80+ cells; blue = 4°-6-diamidino-2-phenylindole [DAPI] and red = F4/80). (B) Representative fluorescence microscopy images show infiltrating CD3+ cells (T lymphocytes) in the perihematomal tissues (the arrows indicate CD3+ cells; blue = DAPI, and green = CD3). (C) CD45+ CD11b+ cells were defined as macrophages and neutrophils and further analyzed to distinguish the two types of cells. (D) The number of macrophages, CD3+ T cells and neutrophils in WT, CCL12-/-, CCL12 overexpression, and vehicle groups after ICH. Scale bars, 50 μm. The bar graphs show the means ± SDs. P-values were determined by ANOVA. *P < 0.05; **P < 0.01; ns, not significant.
Figure 7.  CCL12 antibody treatment improves the prognosis of old mice after ICH. (A-C) NDS (n = 5), mortality rates (B) (n =20) and BWC (C) (n = 5) of old mice treated with an anti-CCL12 antibody after ICH. The bar graphs show the means ± SDs. P-values were determined by ANOVA. *P < 0.05; **P < 0.01; ns, not significant.
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