Application of triple immunohistochemistry to characterize amyloid plaque-associated inflammation in brains with Alzheimer's disease

Inflammation, characterized by the presence of activated microglia and reac tive astrocytes (gliosis), has been described in Alzheimer's disease (AD). We used our routine single immunohistochemical (IHC) labeling protocol to l abel amyloid plaques, an AD neuropathological hallmark, activated microglia , and reactive astrocytes in serial sections of AD hippocampus and entorhin al cortex of brain. Although most amyloid plaques were associated with infl ammation throughout the serial sections, the extent of microglial and astro cytic activation varied among the amyloid plaques. We also observed a popul ation of amyloid plaques that did not appear to coincide with immunolabeled microglia and astrocytes in serial sections, leading us to speculate that some amyloid plaques are not associated with inflammation. Because serial s ectioning limited our ability to confirm these findings, we developed a tri ple IHC protocol to investigate the association of activated microglia and reactive astrocytes simultaneously with amyloid plaques in sections of AD b rain entorhinal cortex and hippocampus. Unlike the potential errors of extr apolating descriptive information from routine IHC or histochemical stainin g methods on sectioned tissues, triple IHC allowed direct characterization of three differently colored antigens in situ. The success of the protocol depended on selection of distinguishable color schemes and resolution of ot her critical technical elements including the compatibility of the reagents and the sensitivity and sequence of the detection systems. The results of the triple IHC protocol clarified the spatial relation of microglia and ast rocytes with amyloid plaques and provoked novel interpretations about the r oles of inflammation in AD brain tissues. We categorized three distinct pop ulations of amyloid plaques related to of inflammation: 1) A beta 42 immuno reactive (a marker of amyloid plaques) amyloid plaques without activated mi croglia or reactive astrocytes, 2) A beta 42-positive amyloid plaques with HLA-DR (a marker of microglia)-positive microglia and no astrocytes, 3) A b eta 42-positive amyloid plaques among HLA-DR and GFAP (a marker of astrocyt es) immunoreactive astrocytes. Most amyloid plaques had varying degrees of activated microglia and reactive astrocytes. Some of the amyloid plaques we re not associated with inflammation while others were associated only with activated microglia. These findings suggest that amyloid plaques without as sociated inflammation may represent recently formed plaques and that the pr esence of amyloid plaques in AD brains may activate microglia prior to glio sis. Furthermore, the shape of the amyloid plaques may be altered subsequen tly from its typical spherical to an aspherical shape by the inflammatory c ells.