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Light Microscopy Approach for Simultaneous Identification of Glial Cells and Amyloid Plaques

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Abstract

The close spatial relationship of glial cells (astroglia and microglia) to amyloid plaques is one of the histopathological features of Alzheimer’s disease. The work aimed to develop a simple and informative method for the simultaneous detection of amyloid plaques and glial cells. The suggested method is based on a combination of histochemical staining for amyloid and non-fluorescent immunohistochemical staining of glial cells. The cerebral cortex samples of aged people (n = 8) and brain samples from transgenic 5×FAD mice (n = 6) were the material for this study. The proposed methodological approach involves immunohistochemical labelling of microglia or astroglia followed by Alcian Blue staining. The developed protocol is simple and well reproducible. It is suitable for identifying the association of glial cells with amyloid plaques. It was noted that immunohistochemistry for Iba1 (ionized calcium binding adaptor molecule 1) and for GFAP (glial fibrillary acidic protein) allows the most effective identification of microgliocytes and astrocytes, respectively. It is also suitable for assessing the functional status of these cells. Amyloid plaques are intensely stained with Alcian Blue and are well detected. In comparison with double immunofluorescence staining technique for simultaneous detection of glial cells and amyloid plaques, the developed method is simple to implement and requires only a light microscope, which makes it accessible to most laboratories.

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REFERENCES

  1. Acosta, C., Anderson, H.D., and Anderson, C.M., Astrocyte dysfunction in Alzheimer disease, J. Neurosci. Res., 2017, vol. 95, p. 2430. https://doi.org/10.1002/jnr.24075

    Article  CAS  PubMed  Google Scholar 

  2. Alekseeva, O.S., Kirik, O.V., Gilerovich, E.G., and Korzhevskii, D.E., Microglia of the brain: origin, structure, functions, J. Evol. Biochem. Physiol., 2019, vol. 55, p. 257. https://doi.org/10.1134/S002209301904001X

    Article  Google Scholar 

  3. Boom, A., Pochet, R., Authelet, M., Pradier, L., Borghgraef, P., Van Leuven, F., Heizmann, C.W., and Brion, J.P., Astrocytic calcium/zinc binding protein S100A6 over expression in Alzheimer’s disease and in PS1/APP transgenic mice models, Biochim. Biophys. Acta, Mol. Cell Res., 2004, vol. 1742, p. 161. https://doi.org/10.1016/j.bbamcr.2004.09.011

    Article  CAS  Google Scholar 

  4. Brozzi, F., Arcuri, C., Giambanco, I., and Donato, R., S100B protein regulates astrocyte shape and migration via interaction with Src kinase, J. Biol. Chem., 2009, vol. 284, p. 8797. https://doi.org/10.1074/jbc.M805897200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Chumasov, E.I., Korzhevskii, D.E., Petrova, E.S., Kuznetsova, N.N., and Sapronov, N.S., Glial reaction of the subventricular zone of the telencephalon of the rat brain on modeling of Alzheimer’s disease, Neurosci. Behav. Physiol., 2012, vol. 42, p. 67. https://doi.org/10.1007/s11055-011-9535-1

    Article  CAS  Google Scholar 

  6. Condello, C., Yuan, P., Schain, A., and Grutzendler, J., Microglia constitute a barrier that prevents neurotoxic protofibrillar Aβ42 hotspots around plaques, Nat. Commun., 2015, vol. 6, p. 6176. https://doi.org/10.1038/ncomms7176

    Article  CAS  PubMed  Google Scholar 

  7. Donato, R., Intracellular and extracellular roles of S100 proteins, Microsc. Res. Techn., 2003, vol. 60, p. 540. https://doi.org/10.1002/jemt.10296

    Article  CAS  Google Scholar 

  8. Dossi, E., Vasile, F., and Rouach, N., Human astrocytes in the diseased brain, Brain Res. Bull., 2018, vol. 136, p. 139. https://doi.org/10.1016/j.brainresbull.2017.02.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Ferrer, I., Diversity of astroglial responses across human neurodegenerative disorders and brain aging, Brain Pathol., 2017, vol. 27, p. 645. https://doi.org/10.1111/bpa.12538

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Fong, A.Y., Stornetta, R.L., Foley, C.M., and Potts, J.T., Immunohistochemical localization of GAD67-expressing neurons and processes in the rat brainstem: subregional distribution in the nucleus tractus solitarius, J. Comp. Neurol., 2005, vol. 493, p. 274. https://doi.org/10.1002/cne.20758

    Article  CAS  PubMed  Google Scholar 

  11. Garaschuk, O. and Verkhratsky, A., GABAergic astrocytes in Alzheimer’s disease, Aging, 2019, vol. 11, p. 1602. https://doi.org/10.18632/aging.101870

    Article  PubMed  PubMed Central  Google Scholar 

  12. Garwood, C.J., Ratcliffe, L.E., Simpson, J.E., Heath, P.R., Ince, P.G., and Wharton, S.B., Review: astrocytes in Alzheimer’s disease and other age-associated dementias: a supporting player with a central role, Neuropathol. Appl. Neurobiol., 2017, vol. 43, p. 281. https://doi.org/10.1111/nan.12338

    Article  CAS  PubMed  Google Scholar 

  13. Guénette, S.Y., Astrocytes: a cellular player in Aβ clearance and degradation, Trends Mol. Med., 2003, vol. 9, p. 279. https://doi.org/10.1016/S1471-4914(03)00112-6

    Article  CAS  PubMed  Google Scholar 

  14. Guselnikova, V., Antipova, M.V., Fedorova, E.A., Safray, A.E., Rukavishnikova, A.A., Mikhailova, E.V., and Korzhevskii, D.E., Distinctive features of histochemical and immunohistochemical techniques for amyloid plaque detection in the human cerebral cortex, J. Anat. Histopathol., 2019, vol. 8, p. 91. https://doi.org/10.18499/2225-7357-2019-8-2-91-99

    Article  Google Scholar 

  15. Habib, N., McCabe, C., Medina, S., Varshavsky, M., Kitsberg, D., Dvir-Szternfeld, R., Green, G., Dionne, D., Nguyen, L., Marshall, J.L., Chen, F., Zhang, F., Kaplan, T., Regev, A., and Schwartz, M., Disease-associated astrocytes in Alzheimer’s disease and aging, Nat. Neurosci., 2020, vol. 23, p. 701. https://doi.org/10.1038/s41593-020-0624-8

    Article  CAS  PubMed  Google Scholar 

  16. Halle, A., Hornung, V., Petzold, G.C., Stewart, C.R., Monks, B.G., Reinheckel, T., Fitzgerald, K.A., Latz, E., Moore, K.J., and Golenbock, D.T., The NALP3 inflammasome is involved in the innate immune response to amyloid-β, Nat. Immunol., 2008, vol. 9, p. 857. https://doi.org/10.1038/ni.1636

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Hansen, D.V, Hanson, J.E., and Sheng, M., Microglia in Alzheimer’s disease, J. Cell Biol., 2018, vol. 217, p. 459. https://doi.org/10.1083/jcb.201709069

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Hopperton, K.E., Mohammad, D., Trépanier, M.O., Giuliano, V., and Bazinet, R.P., Markers of microglia in post-mortem brain samples from patients with Alzheimer’s disease: a systematic review, Mol. Psychiatry, 2018, vol. 23, p. 177. https://doi.org/10.1083/jcb.201709069

    Article  CAS  PubMed  Google Scholar 

  19. Jo, S., Yarishkin, O., Hwang, Y.J., Chun, Y.E., Park, M., Woo, D.H., Bae, J.Y., Kim, T., Lee, J., Chun, H., Park, H.J., Lee, D.Y., Hong, J., Kim, H.Y., Oh, S.J., and et, al., GABA from reactive astrocytes impairs memory in mouse models of Alzheimer’s disease, Nat. Med., 2014, vol. 20, p. 886. https://doi.org/10.1038/nm.3639

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kamphuis, W., Mamber, C., Moeton, M., Kooijman, L., Sluijs, J.A., Jansen, A.H.P., Verveer, M., de Groot, L.R., Smith, V.D., Rangarajan, S., Rodríguez, J.J., Orre, M., and Hol, E.M., GFAP isoforms in adult mouse brain with a focus on neurogenic astrocytes and reactive astrogliosis in mouse models of Alzheimer disease, PLoS One, 2012, vol. 7, article ID e42823. https://doi.org/10.1371/journal.pone.0042823

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Katsouri, L., Birch, A.M., Renziehausen, A.W.J., Zach, C., Aman, Y., Steeds, H., Bonsu, A., Pal-mer, E.O.C., Mirzaei, N., Ries, M., and Sastre, M., Ablation of reactive astrocytes exacerbates disease pathology in a model of Alzheimer’s disease, Glia, 2020, 2020, vol. 68, p. 1017. https://doi.org/10.1002/glia.23759

  22. Kelly, P., Hudry, E., Hou, S.S., and Bacskai, B.J., In vivo two photon imaging of astrocytic structure and function in Alzheimer’s disease, Front. Aging Neurosci., 2018, vol. 10, p. 219. https://doi.org/10.3389/fnagi.2018.00219

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Keren-Shaul, H., Spinrad, A., Weiner, A., Matcovitch-Natan, O., Dvir-Szternfeld, R., Ulland, T.K., David, E., Baruch, K., Lara-Astaiso, D., Toth, B., Itzkovitz, S., Colonna, M., Schwartz, M., and Amit, I., A unique microglia type associated with restricting development of Alzheimer’s disease, Cell, 2017, vol. 169, p. 1276. https://doi.org/10.1016/j.cell.2017.05.018

    Article  CAS  PubMed  Google Scholar 

  24. Kirik, O.V., Sukhorukova, E.G., and Korzhevskii, D.E., Calcium-binding protein Iba-1/AIF-1 in rat brain cells, Neurosci. Behav. Physiol., 2011, vol. 41, p. 149. https://doi.org/10.1007/s11055-011-9391-z

    Article  CAS  Google Scholar 

  25. Korzhevskii, D.E., Kirik, O.V., Petrova, E.S., Karpenko, M.N., Grigoriev, I.P., Sukhorukova, E.G., Kolos, E.A., and Gilyarov, A.V., Teoreticheskie osnovy i prakticheskoe primenenie metodov immunogistokhimii (Theoretical Foundations and Practical Application of Methods of Immunohistochemistry), Korzhevsky, D.E., Ed., St. Petersburg: SpetsLit, 2014, 2nd ed. (rev., add.).

  26. Korzhevskii, D.E., Sukhorukova, E.G., Kirik, O.V., and Grigorev, I.P., Immunohistochemical demonstration of specific antigens in the human brain fixed in zinc-ethanol-formaldehyde, Eur. J. Histochem., 2015, vol. 59, p. 5. https://doi.org/10.4081/ejh.2015.2530

    Article  CAS  Google Scholar 

  27. Korzhevskii, D.E., Grigor’ev, I.P., Gusel’nikova, V.V., Kolos, E.A., Petrova, E.S., Kirik, O.V., Sufieva, D.A., Razenkova, V.A., Antipova, M.V., and Chernysh, M.V., Immunohistochemical markers for neurobiology, Med. Acad. J., 2019, vol. 19, p. 7. https://doi.org/10.17816/MAJ16548

    Article  Google Scholar 

  28. LaFerla, F.M. and Green, K.N., Animal models of Alzheimer disease, Cold Spring Harbor Persp. Med., 2012, vol. 2, article ID a006320. https://doi.org/10.1101/cshperspect.a006320

    Article  CAS  Google Scholar 

  29. Li, K.-Y., Gong, P., Li, J., Xu, N., and Qin, S., Morphological and molecular alterations of reactive astrocytes without proliferation in cerebral cortex of an APP/PS1 transgenic mouse model and Alzheimer’s patients, Glia, 2020, vol. 68, p. 2361. https://doi.org/10.1002/glia.23845

    Article  PubMed  Google Scholar 

  30. Mucke, L., Alzheimer’s disease, Nature, 2009, vol. 461, p. 895. https://doi.org/10.1038/461895a

    Article  CAS  PubMed  Google Scholar 

  31. Nagele, R.G., D’Andrea, M.R., Lee, H., Venkataraman, V., and Wang, H.-Y., Astrocytes accumulate aβ42 and give rise to astrocytic amyloid plaques in Alzheimer disease Brains, Brain Res., 2003, vol. 971, p. 197. https://doi.org/10.1016/S0006-8993(03)02361-8

    Article  CAS  PubMed  Google Scholar 

  32. Nicoll, J.A.R. and Weller, R.O., A new role for astrocytes: β-amyloid homeostasis and degradation, Trends Mol. Med., 2003, vol. 9, p. 281. https://doi.org/10.1016/S1471-4914(03)00109-6

    Article  CAS  PubMed  Google Scholar 

  33. Nosova, O.I., Sufieva, D.A., and Korzhevskii, D.E., An astrocyte structural organization analysis based on fluorescent microscopy with 2D and 3D quantitative approaches, Cell Tissue Biol., 2021, vol. 15, p. 273. https://doi.org/, 2021, vol. 101134/S1990519X21030081.

  34. Oakley, H., Cole, S.L., Logan, S., Maus, E., Shao, P., Craft, J., Guillozet-Bongaarts, A., Ohno, M., Disterhoft, J., Van Eldik, L., Berry, R., and Vassar, R., Intraneuronal beta-amyloid aggregates, meurodegeneration, and meuron loss in transgenic mice with five familial Alzheimer’s disease mutations: potential factors in amyloid plaque formation, J. Neurosci., 2006, vol. 26, p. 10129. https://doi.org/10.1523/JNEUROSCI.1202-06.2006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Olabarria, M., Noristani, H.N., Verkhratsky, A., and Rodríguez, J.J., Concomitant astroglial atrophy and astrogliosis in a triple transgenic animal model of Alzheimer’s disease, Glia, 2010, vol. 58, p. 831. https://doi.org/10.1002/glia.20967

    Article  PubMed  Google Scholar 

  36. Olabarria, M., Noristani, H.N., Verkhratsky, A., and Rodríguez, J.J., Age-dependent decrease in glutamine synthetase expression in the hippocampal astroglia of the triple transgenic Alzheimer’s disease mouse model: mechanism for deficient glutamatergic transmission?, Mol. Neurodegeneration, 2011, vol. 6, p. 55. https://doi.org/10.1186/1750-1326-6-55

    Article  CAS  Google Scholar 

  37. Orellana, J.A., Shoji, K.F., Abudara, V., Ezan, P., Amigou, E., Saez, P.J., Jiang, J.X., Naus, C.C., Saez, J.C., and Giaume, C., Amyloid-induced death in neurons involves glial and neuronal hemichannels, J. Neurosci., 2011, vol. 31, p. 4962. https://doi.org/10.1523/JNEUROSCI.6417-10.2011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Poliakova, A.A., Semernin, E.N., Sitnikova, M.Y., Avagyan, K.L., Grozov, R.V., Pyko, S.A., Krutikov, A.N., Davydova, V.G., Khmelnitskaya, K.A., Shavloskii, M.M., Korzhevskii, D.E., and Gudkova, A.Y., Transthyretin amyloidosis in a cohort of old and very old patients with chronic heart failure, Kardiologiya, 2018, vol. 58, p. 12. https://doi.org/10.18087/cardio.2390

    Article  Google Scholar 

  39. Pomerance, A., Slavin, G., and McWatt, J., Experience with the sodium sulphate-alcian blue stain for amyloid in cardiac pathology, J. Clin. Pathol., 1976, vol. 29, p. 22. https://doi.org/10.1136/jcp.29.1.22

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Razenkova, V.A. and Korzhevskii, D.E., GABAergic axosomatic synapses of rat brain cortex, Cell Tissue Biol., 2021, vol. 15, p. 267. https://doi.org/10.1134/S1990519X21030093

    Article  Google Scholar 

  41. Rodríguez-Arellano, J.J., Parpura, V., Zorec, R., and Verkhratsky, A., Astrocytes in physiological aging and Alzheimer’s disease, Neuroscience, 2016, vol. 323, p. 170. https://doi.org/10.1016/j.neuroscience.2015.01.007

    Article  CAS  PubMed  Google Scholar 

  42. Simpson, J.E., Ince, P.G., Lace, G., Forster, G., Shaw, P.J., Matthews, F., Savva, G., Brayne, C., and Wharton, S.B., Astrocyte phenotype in relation to Alzheimer-type pathology in the ageing brain, Neurobiol. Aging, 2010, vol. 31, p. 578. https://doi.org/10.1016/j.neurobiolaging.2008.05.015

    Article  CAS  PubMed  Google Scholar 

  43. Snow, P., Patel, N., Harrelson, A., and Goodman, C., Neural-specific carbohydrate moiety shared by many surface glycoproteins in drosophila and grasshopper embryos, J. Neurosci., 1987, vol. 7, p. 4137. https://doi.org/10.1523/JNEUROSCI.07-12-04137.1987

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Snow, A.D., Sekiguchi, R.T., Nochlin, D., Kalaria, R.N., and Kimata, K., Heparan sulfate proteoglycan in diffuse plaques of hippocampus but not of cerebellum in Alzheimer’s disease brain, Am. J. Pathol., 1994, vol. 144, p. 337. http://www.ncbi.nlm.nih.gov/pubmed/8311117.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Steiner, J., Bernstein, H.-G., Bielau, H., Berndt, A., Brisch, R., Mawrin, C., Keilhoff, G., and Bogerts, B., Evidence for a wide extra-astrocytic distribution of S100B in human brain, BMC Neurosci., 2007, vol. 8, p. 2. https://doi.org/10.1186/1471-2202-8-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Stepanichev, M.Y., Zdobnova, I.M., Zarubenko, I.I., Lazareva, N.A., and Gulyaeva, N.V., Studies of the effects of central administration of β-amyloid peptide (25–35): pathomorphological changes in the hippocampus and impairment of spatial memory, Neurosci. Behav. Physiol., 2006, vol. 36, p. 101. https://doi.org/10.1007/s11055-005-0167-1

    Article  CAS  PubMed  Google Scholar 

  47. Tzioras, M., Davies, C., Newman, A., Jackson, R., and Spires-Jones, T., APOE at the interface of inflammation, neurodegeneration and pathological protein spread in Alzheimer’s disease, Neuropathol. Appl. Neurobiol., 2019, vol. 45, p. 327. https://doi.org/10.1111/nan.12529

    Article  CAS  PubMed  Google Scholar 

  48. Verkhratsky, A., Olabarria, M., Noristani, H.N., Yeh, C., and Rodriguez, J.J., Astrocytes in Alzheimer’s disease, Neurotherapeutics, 2010, vol. 7, p. 399. https://doi.org/10.1016/j.nurt.2010.05.017

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Verkhratsky, A., Zorec, R., Rodriguez, J.J., and Parpura, V., Pathobiology of neurodegeneration: the role for astroglia, Opera Med. Physiol., 2016, vol. 1, p. 13. https://doi.org/10.20388/OMP2016.001.0019

    Article  PubMed  PubMed Central  Google Scholar 

  50. Verkhratsky, A., Rodrigues, J.J., Pivoriunas, A., Zorec, R., and Semyanov, A., Astroglial atrophy in Alzheimer’s disease, Pflügers Arch., 2019, vol. 471, p. 1247. https://doi.org/10.1007/s00424-019-02310-2

    Article  CAS  PubMed  Google Scholar 

  51. Wu, Z., Guo, Z., Gearing, M., and Chen, G., Tonic inhibition in dentate gyrus impairs long-term potentiation and memory in an Alzheimer’s disease model, Nat. Commun., 2014, vol. 5, p. 4159. https://doi.org/10.1038/ncomms5159

    Article  CAS  PubMed  Google Scholar 

  52. Yeh, C.-Y., Vadhwana, B., Verkhratsky, A., and Rodríguez, J.J., Early astrocytic atrophy in the entorhinal cortex of a triple transgenic animal model of Alzheimer’s disease, ASN Neuro, 2011, vol. 3, p. 272. https://doi.org/10.1042/AN20110025

    Article  CAS  Google Scholar 

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ACKNOWLEDGMENTS

The work was carried out the state assignment of the Institute of Experimental Medicine.

Funding

The work of V.V. Guselnikova was supported by a grant from the President of the Russian Federation to support young russian scientists (MK-560.2020.7).

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  1. Institute of Experimental Medicine, 197376, St. Petersburg, Russia

    O. I. Nosova, V. V. Guselnikova & D. E. Korzhevskii

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  1. O. I. Nosova
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  3. D. E. Korzhevskii
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Correspondence to O. I. Nosova.

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Statement on the welfare of animals. All applicable international and national principles of humane treatment of animals were observed. All the experiments were approved by the Local Ethics Committee of Institute of Experimental Medicine (protocol no. 3/18 dated November 22, 2018).

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Abbreviations: AD – Alzheimer’s disease; GFAP – glial fibrillar acidic protein.

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Nosova, O.I., Guselnikova, V.V. & Korzhevskii, D.E. Light Microscopy Approach for Simultaneous Identification of Glial Cells and Amyloid Plaques. Cell Tiss. Biol. 16, 140–149 (2022). https://doi.org/10.1134/S1990519X22020080

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