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

Abstract

Inflammation, characterized by the presence of activated microglia and reactive astrocytes (gliosis), has been described in Alzheimer's disease (AD). We used our routine single immunohistochemical (IHC) labeling protocol to label amyloid plaques, an AD neuropathological hallmark, activated microglia, and reactive astrocytes in serial sections of AD hippocampus and entorhinal cortex of brain. Although most amyloid plaques were associated with inflammation throughout the serial sections, the extent of microglial and astrocytic activation varied among the amyloid plaques. We also observed a population 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 sectioning limited our ability to confirm these findings, we developed a triple IHC protocol to investigate the association of activated microglia and reactive astrocytes simultaneously with amyloid plaques in sections of AD brain entorhinal cortex and hippocampus. Unlike the potential errors of extrapolating descriptive information from routine IHC or histochemical staining 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 other 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 astrocytes with amyloid plaques and provoked novel interpretations about the roles of inflammation in AD brain tissues. We categorized three distinct populations of amyloid plaques related to of inflammation: 1) Abeta42 immunoreactive (a marker of amyloid plaques) amyloid plaques without activated microglia or reactive astrocytes, 2) Abeta42-positive amyloid plaques with HLA-DR (a marker of microglia)-positive microglia and no astrocytes, 3) Abeta42-positive amyloid plaques among HLA-DR and GFAP (a marker of astrocytes) immunoreactive astrocytes. Most amyloid plaques had varying degrees of activated microglia and reactive astrocytes. Some of the amyloid plaques were not associated with inflammation while others were associated only with activated microglia. These findings suggest that amyloid plaques without associated inflammation may represent recently formed plaques and that the presence of amyloid plaques in AD brains may activate microglia prior to gliosis. Furthermore, the shape of the amyloid plaques may be altered subsequently from its typical spherical to an aspherical shape by the inflammatory cells.

The microglial phagocytic role with specific plaque types in the Alzheimer disease brain

“…Inhibition of microglia was also observed in a separate study where nitric oxide (NO)-related oxidative brain damage was promoted by the microglial cytokine TNF-␣ [41], whereas astrocytes exerted, via the release of TGF-␤, a negative feedback that inhibited microglial NO production [55]. Taken together, these studies suggest that the function of subsequent astrocyte activation may be to temper or regulate the phagocytic microglial activity [16,55,59] perhaps in an effort to more easily form a "scar" of fibrotic processes to heal the necrotic area by providing structural support to the residual tissue like that observed in cases of brain injury involving localized cell death [25]. Similar astrocytic "wraps" were reported in Mongolian gerbils 17 days after transient forebrain ischemia where they formed a structure that was described as a glial scar [18].…”

“…Contrary to the popularized dogma that all amyloid plaques arise from extracellular deposition, plaques may originate from vessels [44], neurons [11][12][13][14][15][16][17], Purkinje cell dendritic processes [67] and astrocytes [46]. In one example, recent attention has turned towards pathological events within the neuron suggesting that cell death could come Fig.…”

“…2. Triple immunohistochemical labeling using specific antibodies to A␤42, HLA-DR and GFAP to detect amyloid, microglia and astrocytes, respectively according to previously published methods [16] from inside the cell, leading to lysis of A␤-filled neurons to deposit their contents as dense-core plaques in the brain [11][12][13]15]. In another example, our lab discovered the presence of astrocytic plaques (lysed astrocytes) in the AD brain tissues [46] thereby supporting the existence of distinctive plaque types in the AD brain.…”

“…1. Triple immunohistochemical labeling using specific antibodies to A␤42, HLA-DR and GFAP to detect amyloid, microglia and astrocytes, respectively according to previously published methods [16] in different areas of the same AD cerebellum. Open arrows indicate red-labeled amyloid plaques.…”

“…Other than released enzymes, cellular nucleotides will also be released, which are diffusible and may play a role in microglial chemotaxis [16,23,27,47]. One such example of microglial-activating material detected in dense-core but not diffuse plaques is a nuclear remnant [11,13,15] as ATP or ADP can induce chemotaxis for cultured microglia through G i/o -coupled P2Y receptors [27], which is completely blocked by either AR-C69931MX, a potent and selective antagonist against P2T AC receptors or pertussis toxin pretreatment [27].…”