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Histochemistry and Cell Biology

, Volume 148, Issue 5, pp 517–528 | Cite as

Progressive accumulation of autofluorescent granules in macrophages in rat striatum after systemic 3-nitropropionic acid: a correlative light- and electron-microscopic study

  • Tae-Ryong Riew
  • Hong Lim Kim
  • Jeong-Heon Choi
  • Xuyan Jin
  • Yoo-Jin Shin
  • Mun-Yong Lee
Original Paper

Abstract

A variety of tissue biomolecules and intracellular structures are known to be autofluorescent. However, autofluorescent signals in brain tissues often confound analysis of the fluorescent markers used for immunohistochemistry. While investigating tissue and cellular pathologies induced by 3-nitropropionic acid, a mitochondrial toxin selective for striatal neurons, we encountered many autofluorescent signals confined to the lesion core. These structures were excited by blue (wavelength = 488 nm) and yellow-orange (555 nm), but not by red (639 nm) or violet (405 nm) lasers, indicating that this autofluorescence overlaps with the emission spectra of commonly used fluorophores. Almost all of the autofluorescence was localized in activated microglia/macrophages, while reactive astrocytes emitted no detectable autofluorescence. Amoeboid brain macrophages filled with autofluorescent granules revealed very weak expression of the microglial marker, ionized calcium-binding adaptor molecule 1 (Iba1), while activated microglia with evident processes and intense Iba1 immunoreactivity contained scant autofluorescent granules. In addition, immunolabeling with two lysosomal markers, ED1/CD68 and lysosomal-associated membrane protein 1, showed a pattern complementary with autofluorescent signals in activated microglia/macrophages, implying that the autofluorescent structures reside within cytoplasm free of intact lysosomes. A correlative light- and electron-microscopic approach finally revealed the ultrastructural identity of the fluorescent granules, most of which matched to clusters of lipofuscin-like inclusions with varying morphology. Thus, autofluorescence in the damaged brain may reflect the presence of lipofuscin-laden brain macrophages, which should be taken into account when verifying any fluorescent signals that are likely to be correlated with activated microglia/macrophages after brain insults.

Keywords

Autofluorescence Lipofuscin 3-NP Microglia Macrophages Lysosome 

Notes

Acknowledgements

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning (NRF-2014R1A2A1A11050246).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. Amenta D, Ferrante F, Franch F, Amenta F (1988) Effects of long-term Hydergine administration on lipofuscin accumulation in senescent rat brain. Gerontology 34(5–6):250–256CrossRefPubMedGoogle Scholar
  2. Aubin JE (1979) Autofluorescence of viable cultured mammalian cells. J Histochem Cytochem Off J Histochem Soc 27(1):36–43CrossRefGoogle Scholar
  3. Banerjee B, Miedema BE, Chandrasekhar HR (1999) Role of basement membrane collagen and elastin in the autofluorescence spectra of the colon. J Investig Med Off Publ Am Feder Clin Res 47(6):326–332Google Scholar
  4. Boellaard JW, Schlote W, Hofer W (2004) Species-specific ultrastructure of neuronal lipofuscin in hippocampus and neocortex of subhuman mammals and humans. Ultrastruct Pathol 28(5–6):341–351. doi: 10.1080/019131290882330 CrossRefPubMedGoogle Scholar
  5. Brizzee KR, Ordy JM, Kaack B (1974) Early appearance and regional differences in intraneuronal and extraneuronal lipofuscin accumulation with age in the brain of a nonhuman primate (Macaca mulatta). J Gerontol 29(4):366–381CrossRefPubMedGoogle Scholar
  6. Brooke SM, Trafton JA, Sapolsky RM (1996) Autofluorescence as a confound in the determination of calcium levels in hippocampal slices using fura-2AM dye. Brain Res 706(2):283–288CrossRefPubMedGoogle Scholar
  7. Brunk UT, Terman A (2002) Lipofuscin: mechanisms of age-related accumulation and influence on cell function. Free Radical Biol Med 33(5):611–619CrossRefGoogle Scholar
  8. Chung YG, Schwartz JA, Gardner CM, Sawaya RE, Jacques SL (1997) Diagnostic potential of laser-induced autofluorescence emission in brain tissue. J Korean Med Sci 12(2):135–142. doi: 10.3346/jkms.1997.12.2.135 CrossRefPubMedPubMedCentralGoogle Scholar
  9. Crespi F, Croce AC, Fiorani S, Masala B, Heidbreder C, Bottiroli G (2004) In vivo autofluorescence spectrofluorometry of central serotonin. J Neurosci Methods 140(1–2):67–73. doi: 10.1016/j.jneumeth.2004.06.019 CrossRefPubMedGoogle Scholar
  10. Croce AC, Spano A, Locatelli D, Barni S, Sciola L, Bottiroli G (1999) Dependence of fibroblast autofluorescence properties on normal and transformed conditions. Role of the metabolic activity. Photochem Photobiol 69(3):364–374CrossRefPubMedGoogle Scholar
  11. Damoiseaux JG, Dopp EA, Calame W, Chao D, MacPherson GG, Dijkstra CD (1994) Rat macrophage lysosomal membrane antigen recognized by monoclonal antibody ED1. Immunology 83(1):140–147PubMedPubMedCentralGoogle Scholar
  12. Davis AS, Richter A, Becker S, Moyer JE, Sandouk A, Skinner J, Taubenberger JK (2014) Characterizing and diminishing autofluorescence in formalin-fixed paraffin-embedded human respiratory tissue. J Histochem Cytochem Off J Histochem Soc 62(6):405–423. doi: 10.1369/0022155414531549 CrossRefGoogle Scholar
  13. Gray DA, Woulfe J (2005) Lipofuscin and aging: a matter of toxic waste. Science of aging knowledge environment: SAGE KE 2005 (5):re1. doi: 10.1126/sageke.2005.5.re1
  14. Hamilton BF, Gould DH (1987) Nature and distribution of brain lesions in rats intoxicated with 3-nitropropionic acid: a type of hypoxic (energy deficient) brain damage. Acta Neuropathol 72(3):286–297CrossRefPubMedGoogle Scholar
  15. Ito D, Imai Y, Ohsawa K, Nakajima K, Fukuuchi Y, Kohsaka S (1998) Microglia-specific localisation of a novel calcium binding protein, Iba1. Brain Res Mol Brain Res 57(1):1–9CrossRefPubMedGoogle Scholar
  16. Jung T, Bader N, Grune T (2007) Lipofuscin: formation, distribution, and metabolic consequences. Ann NY Acad Sci 1119:97–111. doi: 10.1196/annals.1404.008 CrossRefPubMedGoogle Scholar
  17. Kanazawa H, Ohsawa K, Sasaki Y, Kohsaka S, Imai Y (2002) Macrophage/microglia-specific protein Iba1 enhances membrane ruffling and Rac activation via phospholipase C-gamma -dependent pathway. J Biol Chem 277(22):20026–20032. doi: 10.1074/jbc.M109218200 CrossRefPubMedGoogle Scholar
  18. Lei L, Tzekov R, Tang S, Kaushal S (2012) Accumulation and autofluorescence of phagocytized rod outer segment material in macrophages and microglial cells. Molecular vision 18:103–113PubMedPubMedCentralGoogle Scholar
  19. Liu S, Connor J, Peterson S, Shuttleworth CW, Liu KJ (2002) Direct visualization of trapped erythrocytes in rat brain after focal ischemia and reperfusion. J Cereb Blood Flow Metab Off J Int Soc Cereb Blood Flow Metab 22(10):1222–1230. doi: 10.1097/00004647-200210000-00010 CrossRefGoogle Scholar
  20. Markelic M, Velickovic K, Golic I, Klepal W, Otasevic V, Stancic A, Jankovic A, Vucetic M, Buzadzic B, Korac B, Korac A (2013) The origin of lipofuscin in brown adipocytes of hyperinsulinaemic rats: the role of lipid peroxidation and iron. Histol Histopathol 28(4):493–503. doi: 10.14670/hh-28.493 PubMedGoogle Scholar
  21. Mendes-Jorge L, Ramos D, Luppo M, Llombart C, Alexandre-Pires G, Nacher V, Melgarejo V, Correia M, Navarro M, Carretero A, Tafuro S, Rodriguez-Baeza A, Esperanca-Pina JA, Bosch F, Ruberte J (2009) Scavenger function of resident autofluorescent perivascular macrophages and their contribution to the maintenance of the blood–retinal barrier. Invest Ophthalmol Vis Sci 50(12):5997–6005. doi: 10.1167/iovs.09-3515 CrossRefPubMedGoogle Scholar
  22. Miyagishi T, Takahata N, Iizuka R (1967) Electron microscopic studies on the lipo-pigments in the cerebral cortex nerve cells of senile and vitamin E deficient rats. Acta Neuropathol 9(1):7–17CrossRefPubMedGoogle Scholar
  23. Molcanyi M, Bosche B, Kraitsy K, Patz S, Zivcak J, Riess P, El Majdoub F, Hescheler J, Goldbrunner R, Schafer U (2013) Pitfalls and fallacies interfering with correct identification of embryonic stem cells implanted into the brain after experimental traumatic injury. J Neurosci Methods 215(1):60–70. doi: 10.1016/j.jneumeth.2013.02.012 CrossRefPubMedGoogle Scholar
  24. Monici M (2005) Cell and tissue autofluorescence research and diagnostic applications. Biotechnol Ann Rev 11:227–256. doi: 10.1016/s1387-2656(05)11007-2 CrossRefGoogle Scholar
  25. Ohsawa K, Imai Y, Kanazawa H, Sasaki Y, Kohsaka S (2000) Involvement of Iba1 in membrane ruffling and phagocytosis of macrophages/microglia. J Cell Sci 113(Pt 17):3073–3084PubMedGoogle Scholar
  26. Oliveira VC, Carrara RC, Simoes DL, Saggioro FP, Carlotti CG Jr, Covas DT, Neder L (2010) Sudan Black B treatment reduces autofluorescence and improves resolution of in situ hybridization specific fluorescent signals of brain sections. Histol Histopathol 25(8):1017–1024PubMedGoogle Scholar
  27. Ottis P, Koppe K, Onisko B, Dynin I, Arzberger T, Kretzschmar H, Requena JR, Silva CJ, Huston JP, Korth C (2012) Human and rat brain lipofuscin proteome. Proteomics 12(15–16):2445–2454. doi: 10.1002/pmic.201100668 CrossRefPubMedGoogle Scholar
  28. Papka R, Peretz B, Tudor J, Becker J (1981) Age-dependent anatomical changes in an identified neuron in the CNS of Aplysia californica. J Neurobiol 12(5):455–468. doi: 10.1002/neu.480120505 CrossRefPubMedGoogle Scholar
  29. Paxinos G, Watson C (1998) A stereotaxic atlas of the rat brain. Academic, New YorkGoogle Scholar
  30. Robles LJ (1978) Accumulation and identification of lipofuscin-like pigment in the neurons of Bulla gouldiana (Gastropoda: Opisthobranchia). Mech Ageing Dev 7(1):53–64CrossRefPubMedGoogle Scholar
  31. Sasaki Y, Ohsawa K, Kanazawa H, Kohsaka S, Imai Y (2001) Iba1 is an actin-cross-linking protein in macrophages/microglia. Biochem Biophys Res Commun 286(2):292–297. doi: 10.1006/bbrc.2001.5388 CrossRefPubMedGoogle Scholar
  32. Schnell SA, Staines WA, Wessendorf MW (1999) Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue. J Histochem Cytochem Off J Histochem Soc 47(6):719–730CrossRefGoogle Scholar
  33. Spanswick SC, Bray D, Zelinski EL, Sutherland RJ (2009) A novel method for reliable nuclear antibody detection in tissue with high levels of pathology-induced autofluorescence. J Neurosci Methods 185(1):45–49. doi: 10.1016/j.jneumeth.2009.09.007 CrossRefPubMedGoogle Scholar
  34. Spitzer N, Sammons GS, Price EM (2011) Autofluorescent cells in rat brain can be convincing impostors in green fluorescent reporter studies. J Neurosci Methods 197(1):48–55. doi: 10.1016/j.jneumeth.2011.01.029 CrossRefPubMedPubMedCentralGoogle Scholar
  35. Sun Y, Chakrabartty A (2016) Cost-effective elimination of lipofuscin fluorescence from formalin-fixed brain tissue by white phosphor light emitting diode array. Biochem Cell Biol Biochimie et biologie cellulaire 94(6):545–550. doi: 10.1139/bcb-2016-0125 CrossRefPubMedGoogle Scholar
  36. Terman A, Brunk UT (1998) Lipofuscin: mechanisms of formation and increase with age. APMIS: Acta Pathologica, Microbiologica, et Immunologica Scandinavica 106(2):265–276CrossRefPubMedGoogle Scholar
  37. Viegas MS, Martins TC, Seco F, do Carmo A (2007) An improved and cost-effective methodology for the reduction of autofluorescence in direct immunofluorescence studies on formalin-fixed paraffin-embedded tissues. Eur J Histochem EJH 51(1):59–66PubMedGoogle Scholar
  38. Xu H, Chen M, Manivannan A, Lois N, Forrester JV (2008) Age-dependent accumulation of lipofuscin in perivascular and subretinal microglia in experimental mice. Aging Cell 7(1):58–68. doi: 10.1111/j.1474-9726.2007.00351.x CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Tae-Ryong Riew
    • 1
  • Hong Lim Kim
    • 2
  • Jeong-Heon Choi
    • 1
  • Xuyan Jin
    • 1
  • Yoo-Jin Shin
    • 1
  • Mun-Yong Lee
    • 1
  1. 1.Department of Anatomy, Catholic Neuroscience Institute, Cell Death Disease Research Center, College of MedicineThe Catholic University of KoreaSeoulRepublic of Korea
  2. 2.Integrative Research Support Center, Laboratory of Electron Microscope, College of MedicineThe Catholic University of KoreaSeoulRepublic of Korea

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