Distinctive Features of Histochemical and Immunohistochemical Techniques for Amyloid Plaque Detection in the Human Cerebral Cortex

Amyloid plaques (also known as senile plaques) are insoluble extracellular aggregates of the P-amyloid peptide that accumulate in the human brain under normal aging and Alzheimer disease.

The aim of this work was to study distinctive features of amyloid plaque detection in the cerebral cortex of the elderly with histochemical and immunohistochemical techniques.

Material and methods. This study included samples of brain cortex of humans, males and females, aged 79-98, n = 23. Histochemical detection of amyloid was performed using the Congo red and the sodium sulphate-Alcian Blue methods. Anti-amyloid fibrils OC antibodies were used for immunohistochemical identification of amyloid plaques, anti-GFAP antibodies were used for astrocyte detection, a double immunofluorescent OC/GFAP reaction was performed for simultaneous detection of amyloid plaques and astrocytes.

Results. The study results have demonstrated that classical histochemical methods are not effective for identification of amyloid plaques in the human brain allowing detecting only isolated amyloid accumulations. Immunohistochemical reaction with application of anti-amyloid fibrils OC antibodies allows identifying numerous amyloid plaques and studying plaque morphology. Immunohistochemical reaction with application of GFAP, being an astrocyte marker protein, is also suitable for the primary analysis of the human brain samples for the presence or absence of amyloid plaques. The double OC/GFAP immunofluorescence protocol developed by the authors makes it possible to identify both amyloid plaques and astrocytes with high specificity, to further study their spatial relationship and to create 3D reconstructions giving comprehensive information about the structural features and spatial relationship of astrocytes and amyloid plaques.

1. Grigorev IP, Korzhevskii DE. Current Technologies for Fixation of Biological Material for Immunohistochemical Analysis (Review). Sovremennye tehnologii v medicine. 2018 Jun;10(2):156-65 (in Russian).

2. Sukhorukova E.G., Grigor'ev IP, Kirik OV, Korzhevskii DE. Dopolnitel'nye gistologicheskie i immunotsitokhimicheskie metody, ispol'zuemye pri izuchenii neirodegeneratsii. In: Molekulyarnaya neiromorfologiya. Neirodegeneratsiya i otsenka reaktsii nervnykh kletok na povrezhdenie. Saint Petersburg: SpetsLit; 2015 (in Russian). 2

3. Sukhorukova EG, Gusel'nikova VV. Immunogistokhimicheskie markery astrotsitov. In: Immunogistokhimicheskoe issledovanie golovnogo mozga. Saint Petersburg: SpetsLit; 2016 (in Russian).

4. Schavlovsky MM. Ethiology and pathogenesis of amyloidoses: the molecular and genetic basis. Medical Academic Journal. 2010;10(4):63-81 (in Russian).

5. Ariga T, Miyatake T, Yu RK. Role of proteoglycans and glycosaminoglycans in the pathogenesis of Alzheimer’s disease and related disorders: Amyloidogenesis and therapeutic strategies-A review. Journal of Neuroscience Research. 2010 Apr 5;88(11):2303-15. doi: 10.1002/jnr.22393

6. Bennhold H. Specific staining of amyloid with Congo red. Munchener Medizinische Wochenschrifte. 1922; 69:1537-38.

7. Castellani R, Perry G. Molecular Pathology of Alzheimer’s Disease. Colloquium Series on Neurobiology of Alzheimer’s Disease. 2013 Oct 31;1(1):1-91. doi: 10.4199/c00095ed1v01y201310alz001

8. Clement CG, Truong LD. An evaluation of Congo red fluorescence for the diagnosis of amyloidosis. Human Pathology. 2014 Aug;45(8):1766-72. doi: 10.1016/j.humpath.2014.04.016

9. Delaere P, Duyckaerts C, He Y, Piette F, Hauw JJ. Subtypes and differential laminar distributions of?pA4 deposits in Alzheimer’s disease: relationship with the intellectual status of 26 cases. Acta Neuropathologica. 1991 Feb;81(3):328-35. doi: 10.1007/bf00305876

10. Frost GR, Li Y-M. The role of astrocytes in amyloid production and Alzheimer’s disease. Open Biology. 2017 Dec;7(12):170228. doi: 10.1098/rsob.170228

11. Gillmore JD, Hawkins PN. Pathophysiology and treatment of systemic amyloidosis. Nature Reviews Nephrology. 2013 Aug 27;9(10):574-86. doi: 10.1038/nrneph.2013.171

12. Gusel’nikova VV, Antimonova OI, Fedorova EA, Shavlovskii MM, Krutikov AN, Mikhailova EV, et al. Fluorene Derivative Disodium Salt as a New Fluorescent Dye for Identification of Amyloid Deposits in Myocardium of mdx Mice. Cell and Tissue Biology. 2018 May;12(3):213-6. doi: 10.1134/s1990519x18030057

13. Gusel’nikova VV, Antimonova OI, Fedorova EE, Shavlovsky MM, Krutikov AN, Mikhailova EV, et al. Fluorescent characterization of amyloid deposits in the kidneys of mdx mice. European Journal of Histochemistry. 2018 Apr 3;62(2). doi: 10.4081/ejh.2018.2870

14. Gusel’nikova VV, Gudkova AY, Semernin EN, Grudinin NA, Krutikov AN, Shavloskii MM, et al. Characterization of amyloid deposits found in internal organs of mdx mice. Cell and Tissue Biology. 2017 Jan;11(1):27—34. doi: 10.1134/s1990519x17010047

15. Haberland C. Clinical Neuropathology: text and color atlas. New York: Demos Medical; 2007.

16. Ikeda SI, Allsop D, Glenner GG. Morphology and distribution of plaque and related deposits in the brains of Alzheimer's disease and control cases. Lab Invest. 1989 Jan;60:113-22.

17. Kayed R, Head E, Sarsoza F, Saing T, Cotman CW, Necula M, et al. Fibril specific, conformation dependent antibodies recognize a generic epitope common to amyloid fibrils and fibrillar oligomers that is absent in prefibrillar oligomers. Molecular Neurodegeneration. 2007;2(1):18. doi: 10.1186/1750-1326-2-18

18. Kumar A, Singh A, Ekavali. A review on Alzheimer’s disease pathophysiology and its management: an update. Pharmacological Reports. 2015 Apr;67(2):195-203. doi: 10.1016/j.pharep.2014.09.004

19. Liscic RM. Immunological Aspects and AntiAmyloid Strategy for Alzheimer’s Dementia. Archives of Industrial Hygiene and Toxicology. 2013 Dec 1;64(4):603-8. doi: 10.2478/10004-1254-64-2013-2414

20. Masters CL, Bateman R, Blennow K, Rowe CC, Sperling RA, Cummings JL. Alzheimer’s disease. Nature Reviews Disease Primers. 2015 Oct 15;15056. doi: 10.1038/nrdp.2015.56

21. Mavrogiorgou P, Gertz HJ, Ferszt R, Wolf R, Bar KJ, Juckel G. Are routine methods good enough to stain senile plaques and neurofibrillary tangles in different brain regions of demented patients? Psychiatria Danubina. 2011; 23(4): 334-39.

22. Ogomori K, Kitamoto T, Tateishi J, Sato Y, Suetsugu M, Abe M. Beta-protein amyloid is widely distributed in the central nervous system of patients with Alzheimer’s disease. The American journal of pathology [Internet]. 1989 [cited 2019 Apr 17];134(2):243-51. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1879581/

23. Perez-Nievas BG, Serrano-Pozo A. Deciphering the Astrocyte Reaction in Alzheimer’s Disease. Frontiers in Aging Neuroscience. 2018 Apr 25;10. doi: 10.3389/fnagi.2018.00114

24. Perl DP. Neuropathology of Alzheimer’s Disease. Mount Sinai Journal of Medicine: A Journal of Translational and Personalized Medicine. 2010 Jan;77(1):32-42. doi: 10.1002/msj.20157

25. Raghunathan V, Louis D, Wirk B. Gastrointestinal Tract Amyloidosis Presenting With Pneumatosis Intestinalis. Journal of Clinical Medicine Research. 2017;9(7):654-8. doi: 10.14740/jocmr2957w

26. Real de Asua D, Galvan JM, Filigghedu MT, Trujillo D, Costa R, Cadinanos J. Systemic AA amyloidosis: epidemiology, diagnosis, and management. Clinical Epidemiology. 2014 Oct;369-77. doi: 10.2147/clep.s39981

27. Sipe JD, Benson MD, Buxbaum JN, Ikeda S, Merlini G, Saraiva MJM, et al. Amyloid fibril proteins and amyloidosis: chemical identification and clinical classification International Society of Amyloidosis 2016 Nomenclature Guidelines. Amyloid. 2016 Oct;23(4):209-13. doi: 10.1080/13506129.2016.1257986

28. Snow AD, Willmer JP, Kisilevsky R. Sulfated glycosaminoglycans in Alzheimer’s disease. Human Pathology. 1987 May;18(5):506-10. doi: 10.1016/s0046-8177(87)80036-9

29. Wippold FJ, Cairns N, Vo K, Holtzman DM, Morris JC. Neuropathology for the Neuroradiologist: Plaques and Tangles. American Journal of Neuroradiology. 2007 Oct 9;29(1):18-22. doi: 10.3174/ajnr.a0781

30. Yakupova EI, Bobyleva LG, Vikhlyantsev IM, Bobylev AG. Congo Red and amyloids: history and relationship. Bioscience Reports. 2018 Dec 19;39(1):BSR20181415. doi: 10.1042/bsr20181415