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Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain
Published on: February 12, 2016
Immune Cells Promote BDNF Expression by Infiltrated Macrophages via Interleukin 4 in the Cerebral Ischemia of Male
Xiaobo Li1, Yunqian Guan2, Danni Chen3
1Department of Neurology, Northern Jiangsu People's Hospital, Clinical Medical School of Yangzhou University, Yangzhou, China.
Abstract:
We reported that infiltrated Ly6C+ macrophages express brain-derived neurotrophic factor (BDNF) only at the cerebral cortex infarct in a rat dMCAO model. However, the changein neuron-expressed BDNF, the niche components that induce the Ly6C+ cells to express BDNF, and the cellular sources of these components, remain unclear. In this study, immunofluorescence double staining was performed to label BDNF and Ly6C on brain sections at 3, 24, and 48 h following distal middle cerebral artery occlusion (dMCAO) of male rats, and to stain BDNF with Ly6C, IL-4R, and IL-10R. A neutralizing anti-IL-4 antibody was injected into the infarct, and the IL-4 and BDNF concentrations in the subareas of the infarct were determined using enzyme-linked immunosorbent assay. To find out the cellular sources of IL-4, the markers for microglia, T cells, and neurons were co-stained with IL-4 separately. In certain infarct subareas, the main BDNF-expressing cells shifted quickly from NeuN+ neurons to Ly6C+ cells during 24-48 h post-stroke, and the Ly6C+/BDNF+ cells mostly expressed IL-4 receptor. Following IL-4 neutralizing antibody injection, the BDNF, IL-4 protein levels, and BDNF+/Ly6C+ cells decreased significantly. The main IL-4-expressing cell type in this infarct subarea is not neuron either, but immune cells, including microglia, monocyte, macrophages, and T cells. The neurons, maintained BDNF and IL-4 expression in the peri-infarct area. In conclusion, in a specific cerebral subarea of the rat dMCAO model, IL-4 secreted by immune cells is one of the main inducers for Ly6C+ cells to express BDNF.
Insights
Interleukin-4 (IL-4) from immune cells induces Ly6C+ macrophages to produce brain-derived neurotrophic factor (BDNF) in specific brain infarct areas after stroke in rats.
Area of Science:
- Neuroscience
- Immunology
- Stroke Research
Background:
- Ly6C+ macrophages express brain-derived neurotrophic factor (BDNF) in cerebral cortex infarcts following distal middle cerebral artery occlusion (dMCAO) in rats.
- The specific niche components inducing Ly6C+ cells to express BDNF and their cellular sources remain unclear.
Purpose of the Study:
- To investigate the dynamic changes in neuron-expressed BDNF.
- To identify niche components that induce Ly6C+ cells to express BDNF.
- To determine the cellular sources of these inducing components in a rat dMCAO model.
Main Methods:
- Immunofluorescence double staining for BDNF, Ly6C, IL-4 receptor (IL-4R), and IL-10 receptor (IL-10R) at 3, 24, and 48 hours post-dMCAO.
- Injection of neutralizing anti-IL-4 antibody into the infarct.
- Enzyme-linked immunosorbent assay (ELISA) to measure IL-4 and BDNF concentrations.
- Co-staining for IL-4 with markers for microglia, T cells, and neurons.
Main Results:
- BDNF expression shifted from neurons to Ly6C+ cells within 24-48 hours post-stroke in specific infarct subareas.
- Ly6C+/BDNF+ cells predominantly expressed IL-4R.
- Neutralizing IL-4 significantly reduced BDNF and IL-4 levels, and the number of BDNF+/Ly6C+ cells.
- Immune cells (microglia, monocytes, macrophages, T cells) were identified as the main IL-4 producers in the infarct, not neurons.
- Neurons in the peri-infarct area maintained BDNF and IL-4 expression.
Conclusions:
- Interleukin-4 (IL-4) secreted by immune cells is a key inducer for Ly6C+ cells to express BDNF in specific cerebral subareas following dMCAO in rats.
- This finding elucidates a novel immune-mediated mechanism regulating neurotrophic factor expression in the stroke-affected brain.
