Quantitative immunohistochemical analysis of myeloid cell marker expression in human cortex captures microglia
Molly E V Swanson1,2, Helen C Murray1,2, Brigid Ryan1,2
1Department of Anatomy and Medical Imaging, Faculty of Medical and Health Science, University of Auckland, Private Bag 92019, Auckland, New Zealand.
Insights
Investigating microglial heterogeneity in post-mortem human brain tissue using immunohistochemistry reveals distinct functional states. This approach integrates protein expression and cell morphology to better understand microglial responses to damage and disease.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Current immunohistochemistry methods for human post-mortem brain microglia lack detail on functional heterogeneity.
- Microglial function is critical in neurological damage and disease, but current methods do not fully capture this complexity.
Purpose of the Study:
- To investigate microglial heterogeneity in post-mortem human brain tissue.
- To explore the expression of myeloid cell proteins associated with functional changes in microglia.
- To correlate protein expression and cell morphology for inferring microglial function.
Main Methods:
- Immunohistochemistry on post-mortem human middle temporal gyrus sections.
- Investigated co-expression of HLA-DR with CD206, CD32, CD163, and L-Ferritin.
- Qualitative assessment of protein immunoreactivity and microglial morphology.
Main Results:
- Significant co-labelling was observed between HLA-DR and CD206, CD32, CD163, and L-Ferritin, but not complete overlap.
- Perivascular macrophages showed higher expression of investigated markers than microglia, indicating a more phagocytic and antigen-presenting state.
- L-Ferritin, a marker of degeneration, was specific to dystrophic microglia.
Conclusions:
- Microglial heterogeneity in post-mortem human brain tissue can be studied by integrating protein abundance and cell morphology.
- This integrated approach allows for inference of microglial function in response to neurological conditions.
- The findings provide a foundation for more nuanced studies of microglial roles in brain health and disease.
Abstract:
Current immunohistochemical methods of studying microglia in the post-mortem human brain do not capture the heterogeneity of microglial function in response to damage and disease. We therefore investigated the expression of eight myeloid cell proteins associated with changes in function alongside Iba1. To study the myeloid cells we used immunohistochemistry on post-mortem human middle temporal gyrus sections from neurologically normal individuals. First we investigated co-labelling between the classical 'activation' marker, HLA-DR and each of the other markers of interest. Significant co-labelling between HLA-DR with CD206, CD32, CD163, or L-Ferritin was observed, although complete overlap of expression of HLA-DR with aforementioned markers was not observed. A qualitative assessment also demonstrated that perivascular macrophages expressed higher levels of the markers of interest we investigated than microglia, suggesting perivascular macrophages show a more phagocytic and antigen presentation state in the human brain. To determine whether the markers of interest were expressed in different functional states, the immunoreactivity for each marker was qualitatively assessed on microglial morphologies. Degenerating marker, L-Ferritin, was specific for dystrophic microglia. We demonstrate that microglial heterogeneity can be investigated in immunohistochemically stain post-mortem human tissue by integrating the single-cell abundance of proteins and cell morphology to infer function.
