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01.12.2017 | Research | Ausgabe 1/2017 Open Access

Journal of Neuroinflammation 1/2017

Infiltration and persistence of lymphocytes during late-stage cerebral ischemia in middle cerebral artery occlusion and photothrombotic stroke models

Journal of Neuroinflammation > Ausgabe 1/2017
Yan Feng, Shiwei Liao, Changjuan Wei, Dongmei Jia, Kristofer Wood, Qiang Liu, Xiaoying Wang, Fu-Dong Shi, Wei-Na Jin
Wichtige Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s12974-017-1017-0) contains supplementary material, which is available to authorized users.



Evidence suggests that brain infiltration of lymphocytes contributes to acute neural injury after cerebral ischemia. However, the spatio-temporal dynamics of brain-infiltrating lymphocytes during the late stage after cerebral ischemia remains unclear.


C57BL/6 (B6) mice were subjected to sham, photothrombosis, or 60-min transient middle cerebral artery occlusion (MCAO) procedures. Infarct volume, neurodeficits, production of reactive oxygen species (ROS) and inflammatory factors, brain-infiltrating lymphocytes, and their activation as well as pro-inflammatory cytokine IFN-γ production were assessed. Brain-infiltrating lymphocytes were also measured in tissue sections from post-mortem patients after ischemic stroke by immunostaining.


In mice subjected to transient MCAO or photothrombotic stroke, we found that lymphocyte infiltration persists in the ischemic brain until at least day 14 after surgery, during which brain infarct volume significantly diminished. These brain-infiltrating lymphocytes express activation marker CD69 and produce proinflammatory cytokines such as IFN-γ, accompanied with a sustained increase of reactive oxygen species (ROS) and inflammatory cytokines release in the brain. In addition, brain-infiltrating lymphocytes were observed in post-mortem brain sections from patients during the late stage of ischemic stroke.


Our results demonstrate that brain-infiltration of lymphocytes persists after the acute stage of cerebral ischemia, facilitating future advanced studies to reveal the precise role of lymphocytes during late stage of stroke.
Additional file 1: Microglial phenotype at day 14 after photothrombosis and MCAO. C57BL/6 (B6) mice were subjected to sham operation, photothrombosis, or 60 mins MCAO for 14 days. At day 14 after surgery, mice were subjected to flow cytometry assessment. (A) Dot plots of flow cytometry assay show the gating strategy for microglia and CD68-expression. (B) Bar graphs summarize the cumulative data for quantifying microglia population and CD68-expression from brains of photothrombosis- or MCAO-operated mice at 14 days after stroke. n = 4 mice per group. *P < 0.05; **P < 0.01, sham vs. stroke (photothrombosis/MCAO) by one-way ANOVA. (DOCX 84 kb)
Additional file 2: Lymphocytes infiltration at day 14 after brain ischemia in ET-1 model. (A) ET-1 model induction. ET-1 and L-NAME were dissolved in sterile saline, and were delivered into the cortex by stereotaxic injection (AP +1.0, ML +1.0, DV -1.0, ET-1 at 1 μg and L-NAME at 2.7 μg). The ipsilateral common carotid artery were permanently occluded just prior to the ET-1 injection. (B) Lymphocyte infiltration at late stage of brain ischemia in mice subjected to ET-1 model. Dot plots of flow cytometry assay show CD4+ T, CD8+ T, NK, and B cells in single cell suspension from brains of sham (PBS injection) or ET-1/L-NAME injected mice at 14 days after procedures. (C) Bar graphs summarize the cumulative data for quantifying CD4+ T, CD8+ T, NK, and B cell counts from brains of ET-1 model at 14 days after stroke. n = 8 mice per group. Error bars represent s.e.m.; *P < 0.05; **P < 0.01, sham vs. ET-1 model by two-tailed unpaired Student’s t test. (DOCX 298 kb)
Additional file 3: ROS generation and lymphocytes infiltration in photothrombosis model with lymphocytes pre- or post-stroke transfer. Lymphocytes were isolated from spleens of C57BL/6 mice. 2 × 107 isolated cells were then adoptively transferred into Rag2−/−γc−/− mice followed by sham or photothrombosis procedures, or transferred immediately after procedure. (A) Imaging ROS activity in vivo. Bioluminescent images were captured for 1 min using the cooled IVIS imaging system (Xenogen IVIS-200) after luminol i.p. injection, to monitor the ROS generation in Rag2−/−γc−/− photothrombosis brains. (B) Quantification and statistical analysis of the images. As compared to the pre-stroke cell transfer, post-stroke transferred mice exhibit slightly fewer ROS signal but with no significance. n = 3 mice per group. (C) Lymphocytes were isolated from spleens of C57BL/6 (B6) mice and co-cultured with MIRB. 2 × 107 MIRB-labeled cells were then adoptively transferred into Rag2−/−γc−/− mice followed by sham or stroke procedures, or transferred immediately after procedure. MRI was used to track MIRB-labeled lymphocytes in the ischemic brains of Rag2−/−γc−/− mice. Bar graph shows MIRB signal in mice receiving either sham or photothrombosis model. MIRB signals can be observed in the ischemic brain of post-stroke transferred mice, with comparable intensity to the pre-transferred group. Error bars represent s.e.m.; *P < 0.05; **P < 0.01, sham vs. stroke by one-way ANOVA. (DOCX 257 kb)
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