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Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain
Published on: February 12, 2016
Splenic atrophy in experimental stroke is accompanied by increased regulatory T cells and circulating macrophages
Halina Offner1, Sandhya Subramanian, Susan M Parker
1Neuroimmunology Research, Veterans Affairs Medical Center, Portland, OR 97239, USA. offnerva@ohsu.edu
Insights
Stroke induces splenic atrophy and immunosuppression, characterized by cell death and increased regulatory T cells. This brain injury response reduces peripheral immune function and inflammatory factors in the brain.
Area of Science:
- Neuroscience
- Immunology
- Pathology
Background:
- Stroke causes local brain damage and affects peripheral immunity.
- Peripheral immune responses following stroke are not fully understood.
Purpose of the Study:
- To evaluate the effects of stroke on spleen and blood cells 4 days post-induction.
- To investigate the mechanisms of stroke-induced immunosuppression.
Main Methods:
- Middle cerebral artery occlusion (MCAO) model in rodents.
- Flow cytometry and TUNEL assay to assess cell apoptosis and populations.
- Analysis of cytokine profiles in brain tissue.
Main Results:
- Stroke induced splenic atrophy with reduced splenocyte numbers and increased apoptosis.
- Significant reduction in T cell proliferation and inflammatory cytokine secretion.
- Decreased B cell numbers and increased CD4+FoxP3+ regulatory T cells in spleen and blood.
- Increased nonapoptotic CD11b+ VLA-4-negative macrophages/monocytes in blood.
Conclusions:
- Stroke triggers profound peripheral immunosuppression via splenic cell death and regulatory T cell expansion.
- This immunosuppression may reduce brain inflammation but also impair protective immune responses.
- Brain injury sends a potent negative signal to the peripheral immune system.
Abstract:
Induction of stroke not only produces local ischemia and brain damage, but also has profound effects on peripheral immune responses. In the current study, we evaluated effects on spleen and blood cells 4 days after stroke induction. Surprisingly, there was a less inflammatory cytokine profile in the middle cerebral artery occlusion-affected right brain hemisphere at 96 h compared with earlier time points. Moreover, our results demonstrate that stroke leads to splenic atrophy characterized by a reduction in organ size, a drastic loss of splenocyte numbers, and induction of annexin V+ and TUNEL+ cells within the spleen that are in the late stages of apoptosis. The consequence of this process was to reduce T cell proliferation responses and secretion of inflammatory cytokines, resulting in a state of profound immunosuppression. These changes produced a drastic reduction in B cell numbers in spleen and blood, and a novel increase in CD4+FoxP3+ regulatory T cells. Moreover, we detected a striking increase in the percentage of nonapoptotic CD11b+ VLA-4-negative macrophages/monocytes in blood. Immunosuppression in response to brain injury may account for the reduction of inflammatory factors in the stroke-affected brain, but also potentially could curtail protective immune responses in the periphery. These findings provide new evidence to support the contention that damage to the brain caused by cerebral ischemia provides a powerful negative signal to the peripheral immune system that ultimately induces a drastic state of immunosuppression caused by cell death as well as an increased presence of CD4+FoxP3+ regulatory T cells.
