Fig 1: Immunohistochemistry of inflammatory cells in DRG of normal controls and FA. a-b normal controls; c-d FA; a and c CD68; b and d IBA1. a and b show the relative abundance of CD68- and IBA1-immunoreactive cells in normal DRG. Monocytes abut normal neurons (N) ([a and b], insets) but do not penetrate into the neuronal cytoplasm. In FA, a neuron shows invasion by CD68-positive monocytes resembling neuronophagia (inset) (c). Multiple IBA1-positive monocytes lie immediately adjacent to a degenerating nerve cell (N) (d). One ameboid monocyte appears to project thin processes along and through the neuronal plasma membrane (d, inset). Bars: (a-d), 100 μm; (a) and (c), insets, 20 μm; (b) and (d), insets, 10 μm
Fig 2: Microglia-associated osteopontin levels are significantly increased in HAND and ALS. See description of quantification scheme given in figure legend 2. The results are expressed as the ratio of a OPN to Iba1/AIF-1 or b Iba1/AIF-1 to OPN. One-way ANOVA, with Tukey’s correction for multiple comparisons and significance of p < 0.05, was determined with GraphPad Prism 6. Normal, ALS, amyotrophic lateral sclerosis, HIV-infected cases asymptomatic neurocognitive disorder (HIV+ ANI), and HIV-infected cases with minor neurocognitive disorder/HIV-associated dementia (HIV+ MND/HAD); ****p < 0.0001
Fig 3: Osteopontin (OPN) expression in Iba1/AIF-1+ microglia. See staining paradigm as given in figure legend 1. a case 16, normal; b case 4, normal; c case 29, ALS; d case 23, ANI; e case 12, MND; f case 13, HAD
Fig 4: Double-label immunofluorescence of IBA1 and S100; and IBA1 and cytosolic ferritin in DRG of normal controls and FA. a-b normal controls; c-d FA. a and c double-label immunofluorescence of S100 (Alexa Fluor 488 green) and IBA1 (Cy3 red); DAPI (blue); b and d double-label immunofluorescence of IBA1 (Alexa Fluor 488 green) and cytosolic ferritin (Cy3 red). In the normal DRG, IBA1-reactive monocytes may gain access to the S100-reactive satellite cell sheath around neurons (N) but remain separated from the neuronal plasma membrane by a thin layer of satellite cell cytoplasm (a, arrow). IBA1 and S100 show no colocalization. In FA, the outline of the S100-positive satellite cell layer appears irregular and disrupted. An IBA1-reactive monocyte (arrow) abuts or penetrates the neuronal plasma membrane (c). The inset in (c) shows infiltration of a residual nodule by IBA1-positive monocytes. DAPI fluorescence in (c) confirms increased cellularity in FA when compared to the normal DRG (a). A normal DRG shows ferritin immunofluorescence in satellite cells and neurons (N) (b). The mixed yellow and green color suggests that ferritin biosynthesis also occurs in IBA1-positive monocytes. In FA, a multilayered rim of satellite cells around a shrunken neuron (N) is intensely fluorescent for cytosolic ferritin (Cy3 red) (d). Ameboid monocytes express both ferritin and IBA1. The inset in (d) shows matching single-color images in further support of the colocalization of IBA1 (Alexa Fluor 488 green) and ferritin (Cy3 red). Bars (all): 20 μm
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