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.
Highlights
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
Immunoreactivity for P2RY12, TMEM119, human leukocyte antigen-DR isotype (HLA-DR), CD74, CD206, CD32, CD163, and L-Ferritin was observed on Iba1-positive cells (Fig. 1)
We carried out this quantification in both the grey matter (GM) and white matter (WM) as previous research has not investigated the potential differences in the markers of interest (MOIs) expression between these regions
Summary
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 investigated the expression of these myeloid cell proteins in the human brain by co-labelling them with the classical immunohistochemical ‘activation’ marker, human leukocyte antigen-DR isotype (HLA-DR), investigating their expression on microglia and perivascular macrophages, and identifying their expression on microglia with different morphologies. HLA-DR is an antigen presentation molecule involved in the activation of the adaptive immune system This function, along with high HLA-DR immunoreactivity being identified surrounding amyloid beta plaques in human Alzheimer’s disease brains, resulted in HLA-DR being classed as a marker of pan microglial ‘activation’ in immunohistochemistry studies[30,31]. This presents challenges when considering the heterogeneity of microglial reactions to damage and disease. Determining the expression of this subset of proteins relative to the classical immunohistochemical activation marker allowed us to determine whether they are expressed by unique populations of myeloid cells that do not express HLA-DR
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