The application of alcian blue to identify astrocyte-associated amyloid plaques by using fluorescence and confocal microscopy.

BACKGROUND: Astrocytes play an essential role in the normal functioning of the nervous system and are active contributors to the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD). Therefore, to comprehend the astrocytes and amyloid plaques relationship there is a need for imaging techniques providing simultaneous visualization of astrocytes using fluorescence and amyloid plaques revealed by transmitted light microscopy. NEW METHOD: The possibility of simultaneous detection of astrocytes by immunocytochemistry (fluorescent) and amyloid plaques by cytochemical Alcian Blue (transparent) using confocal microscopy in 8-month-old 5хFAD mice samples shown. RESULTS: The described method supposes performing astrocytes fluorescent labelling by GFAP or S100beta and amyloid plaques staining by Alcian Blue. COMPARISON WITH EXISTING METHODS: Proposed approach circumvents some limitations of fluorescence microscopy, such as weak fluorescence, low contrast, fluorophore broad excitation/emission profile and chemical instability. CONCLUSIONS: The proposed technique provides high-quality resulting images of GFAP/s100beta- labelled astrocytes and Alcian Blue-stained amyloid plaques. These images are appliable for prospective qualitative and quantitative three-dimensional analysis due to the z-axis scanning. Moreover, it demonstrated the formation of stable Alcian Blue staining.

Changes in cytoplasmic and extracellular neuromelanin in human substantia nigra with normal aging.

Neuromelanin (NM) is a dark polymer pigment produced in certain populations of catecholaminergic neurons in the brain. It is present in various areas of the human brain, most often in the substantia nigra (SN) pars compacta and the locus coeruleus, the main centers of dopaminergic and noradrenergic innervation, respectively. Interest in NM has revived in recent years due to the alleged link between NM and the particular vulnerability of neuromelanin-containing neurons to neurodegeneration. The aim of this work was to study the structural, cytochemical, and localization features of cytoplasmic and extracellular neuromelanin in the human SN pars compacta during normal aging. Sections of human SN from young/middle-aged adults (25 to 51 years old, n=7) and older adults (60 to 78 years old, n=5), all of which had no neurological disorders, were stained histochemically for metals (Perls' reaction, Mayer's hematoxylin) and immunohistochemically for tyrosine hydroxylase (TH) and Iba-1. It was shown that dopaminergic neurons in SN pars compacta differ in the amount of neuromelanin and the intensity of TH-immunoreactivity. The number of neuromelanin-containing neurons with decreased TH-immunoreactivity positively correlates with age. Extracellular NM is present in SN pars compacta in both young/middle-aged and older adults. The number of extracellular NM accumulations increases with aging. Cytoplasmic and extracellular NM are predominantly not stained using histochemical methods for detecting metals in people of all ages. We did not detect the appearance of amoeboid microglia in human SN pars compacta with aging, but we found an age-related increase in microglial phagocytic activity. The absence of pronounced microgliosis, as well as a pronounced loss of neuromelanin-containing neurons, indicate the absence of neuroinflammation in human SN pars compacta during normal aging.

Study of Antibody-Dependent Reactions of Mast Cells In Vitro and in a Model of Severe Influenza Infection in Mice.

We investigated the reaction of mouse peritoneal mast cells (MCs) in vitro after IgG-containing immune complex introduction using A/H5N1 and A/H1N1pdm09 influenza viruses as antigens. The sera of immune mice served as a source of IgG antibodies. The concentration of histamine in the supernatants was determined at 4 hours after incubation with antisera and virus. We compared the contribution of MCs to the pathogenesis of post-immunization influenza infection with A/H5N1 and A/H1N1 influenza viruses in mice. The mice were immunized parenterally with inactivated viruses and challenged with lethal doses of drift A/H5N1 and A/H1N1 influenza viruses on the 14th day after immunization. Simultaneously, half of the mice were injected intraperitoneally with a mixture of histamine receptor blockers (chloropyramine and quamatel). In in vitro experiments, the immune complex formed by A/H5N1 virus and antiserum caused a significant increase in the histamine release compared to immune serum or the virus alone. With regard to the A/H1N1 virus, such an increase was not significant. A/H1N1 immunization caused detectable HI response in mice at 12th day after immunization, in contrast to the A/H5N1 virus. After challenge of A/H5N1-immunized mice, administration of antihistamines increased the survival rate by up to 90%. When infecting the A/H1N1-immunized mice, 90% of the animals were already protected from lethal infection by day 14; the administration of histamine receptor blockers did not increase survival. Histological examination of the lungs has shown that toluidine blue staining allows to estimate the degree of MC degranulation. The possibility of in vitro activation of murine MCs by IgG-containing immune complexes has been shown. In a model of influenza infection, it was shown that the administration of histamine receptor blockers increased survival. When the protection was formed faster due to the earlier production of HI antibodies, the administration of histamine receptor blockers did not significantly affect the course of the infection. These data allow to propose that even if there are antibody-dependent MC reactions, they can be easily stopped by the administration of histamine receptor blockers.

Meningeal Mast Cells Contribute to ATP-Induced Nociceptive Firing in Trigeminal Nerve Terminals: Direct and Indirect Purinergic Mechanisms Triggering Migraine Pain.

Peripheral mechanisms of primary headaches such as a migraine remain unclear. Meningeal afferents surrounded by multiple mast cells have been suggested as a major source of migraine pain. Extracellular ATP released during migraine attacks is a likely candidate for activating meningeal afferents via neuronal P2X receptors. Recently, we showed that ATP also increased degranulation of resident meningeal mast cells (Nurkhametova et al., 2019). However, the contribution of ATP-induced mast cell degranulation in aggravating the migraine pain remains unknown. Here we explored the role of meningeal mast cells in the pro-nociceptive effects of extracellular ATP. The impact of mast cells on ATP mediated activation of peripheral branches of trigeminal nerves was measured electrophysiologically in the dura mater of adult wild type (WT) or mast cell deficient mice. We found that a spontaneous spiking activity in the meningeal afferents, at baseline level, did not differ in two groups. However, in WT mice, meningeal application of ATP dramatically (24.6-fold) increased nociceptive firing, peaking at frequencies around 10 Hz. In contrast, in mast cell deficient animals, ATP-induced excitation was significantly weaker (3.5-fold). Application of serotonin to meninges in WT induced strong spiking. Moreover, in WT mice, the 5-HT3 antagonist MDL-7222 inhibited not only serotonin but also the ATP induced nociceptive firing. Our data suggest that extracellular ATP activates nociceptive firing in meningeal trigeminal afferents via amplified degranulation of resident mast cells in addition to direct excitatory action on the nerve terminals. This highlights the importance of mast cell degranulation via extracellular ATP, in aggravating the migraine pain.

Activation of P2X7 Receptors in Peritoneal and Meningeal Mast Cells Detected by Uptake of Organic Dyes: Possible Purinergic Triggers of Neuroinflammation in Meninges.

Extracellular ATP activates inflammasome and triggers the release of multiple cytokines in various immune cells, a process primarily mediated by P2X7 receptors. However, the expression and functional properties of P2X7 receptors in native mast cells in tissues such as meninges where migraine pain originates from have not been explored. Here we report a novel model of murine cultured meningeal mast cells and using these, as well as easily accessible peritoneal mast cells, studied the mechanisms of ATP-mediated mast cell activation. We show that ATP induced a time and dose-dependent activation of peritoneal mast cells as analyzed by the uptake of organic dye YO-PRO1 as well as 4,6-diamidino-2-phenylindole (DAPI). Both YO-PRO1 and DAPI uptake in mast cells was mediated by the P2X7 subtype of ATP receptors as demonstrated by the inhibitory effect of P2X7 antagonist A839977. Consistent with this, significant YO-PRO1 uptake was promoted by the P2X7 agonist 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP). Extracellular ATP-induced degranulation of native and cultured meningeal mast cells was shown with Toluidine Blue staining. Taken together, these data demonstrate the important contribution of P2X7 receptors to ATP-driven activation of mast cells, suggesting these purinergic mechanisms as potential triggers of neuroinflammation and pain sensitization in migraine.