Patterns of Apoptosis and Autophagy Activation After Hydroxyurea Exposure in the Rat Cerebellar External Granular Layer: an Immunoperoxidase and Ultrastructural Analysis

  • Vanessa Molina
  • Lucía Rodríguez-Vázquez
  • Joaquín MartíEmail author
Original Article


The time courses of apoptosis and autophagy activation were investigated in neuroblasts of the cerebellar external granular layer (EGL) following the treatment with a single dose (2 mg/g) of hydroxyurea (HU), a cytotoxic agent. The rats were examined at postnatal day 9 and sacrificed at appropriate times ranging from 10 to 60 h after drug administration. We used the Feulgen method, the TUNEL assay, immunohistochemistry for active caspase-3, and LC3B and p62/SQSTM1 immunoperoxidase procedures. The resulting data indicated that the administration of HU leads to the activation of apoptotic cellular events that began to increase 10 h after HU exposure, peaked at 30 h, and decrease thereafter. It also showed that apoptosis was followed by autophagy activation. Interestingly, LC3B and p62/SQSTM1-stained cells, as well as mitotic cells, started to appear 20 h after the HU injection and their counts increased until 40 h. Afterwards, the values remained stable. The current results highlight an important role of the apoptotic and autophagic processes in the EGL after HU administration. Moreover, they provide a clue for studying the mechanism of chemoresistance triggered by proliferating cells exposed to anticancer agents.


Perinatal Cerebellar cortex External granular layer Hydroxyurea Apoptosis Autophagy Electron microscopy 



The authors are very grateful to Drs. María del Carmen Santa-Cruz and José Pablo Hervás for providing the animals.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflicts of interest.


  1. Altman J, Bayer SA (1997) Development of the cerebellar system: in relation to its evolution, structure and functions. CRC Press, Inc, Boca RatonGoogle Scholar
  2. Alvarez-Larran A, Kerguelen A, Hernández-Boluda JC, Pérez-Encinas M, Ferrer-Marín F, Bárez A, Martínez-López J, Cuevas B, Mata MI, García-Gutierrez V, Aragües P, Motesdeoca S, Burgaleta C, Caballero G, Hernández-Rivas JA, Duran MA, Gómez-Casares MT (2016) Besses C, on the behalf of the grupo español de enfermedades mieloproliferativas Filadelfia negativas. Br J Haematol 172:786–793CrossRefGoogle Scholar
  3. Berezowska S, Galván JA (2017) Immunohistochemical detection of the autophagy markers LC3 and p62/SQSTM1 in formalin-fixed and paraffin-embedded tissue. Methods Mol Biol 1560:189–194CrossRefGoogle Scholar
  4. Butts T, Green MJ, Wingate RJ (2014) Development of the cerebellum: simple steps to make a ‘little brain’. Development 141:4031–4041CrossRefGoogle Scholar
  5. Chédotal A (2010) Should I stay or should I go? Becoming a granule cell. Trends Neurosci 33:163–172CrossRefGoogle Scholar
  6. Doi K (2011) Mechanisms of neurotoxicity induced in the developing brain of mice and rats by DNA-damaging chemicals. J Toxicol Sci 36(6):695–712CrossRefGoogle Scholar
  7. Ebels EJ, Peters I, Thijs A (1975) Studies on ectopic granule cells in the cerebellar cortex. III. An investigation into the restoration of the external granular layer after partial destruction. Acta Neuropathol 31:103–107CrossRefGoogle Scholar
  8. Erekat NS (2018) Autophagy precedes apoptosis among at risk cerebellar Purkinje cells in the shaker mutant rat: an ultrastructural study. Ultrastruct Pathol 42:162–169CrossRefGoogle Scholar
  9. Eskelinen EL (2008) To be or not to be? Examples of incorrect identification of autophagic compartments in conventional transmission electron microscopy of mammalian cells. Autophagy 4:257–260CrossRefGoogle Scholar
  10. Eskelinen EL, Reggiori F, Baba M, Kovács AL, Seglen PO (2011) Seeing is believing: the impact of electron microscopy on autophagy research. Autophagy 7:935–956CrossRefGoogle Scholar
  11. Fang X, Yin H, Zhang H, Wu F, Liu Y, Fu Y, Yu D, Zong L (2019) p53 mediates hydroxyurea resistance in aneuploid of colon cancer. Exp Cell Res 376:39–48CrossRefGoogle Scholar
  12. Fricker M, Tolkovsky AM, Borutaite V, Coleman M, Brown GC (2018) Neuronal cell death. Physiol Rev 98:813–880CrossRefGoogle Scholar
  13. Ghavami S, Shojaei S, Yeganeh B, Ande SR, Jangamreddy JR, Mehrpour M, Christoffersson J, Chaabane W, Moghadam AR, Kashani HH, Hashemi M, Owji AA, Los MJ (2014) Autophagy and apoptosis dysfunction in neurodegenerative disorders. Prog Neurobiol 112:24–49CrossRefGoogle Scholar
  14. Girault V, Gilard V, Marquet F, Lesueur C, Hauchecorne M, Ramdani Y, Laquerrière A, Marret S, Jégou S, Gonzalez BJ, Brasse-Lagnel C, Bekro S (2017) Prenatal alcohol exposure impairs autophagy in neonatal brain cortical microvessels. Cell Death Dis 8(2):e2610CrossRefGoogle Scholar
  15. Grimaldi M, Dal Bo V, Ferrari B, Roda E, De Luca F, Veneroni P, Barni S, Verri M, De Pascali SA, Fanizzi FP, Bernocchi G, Bottone MG (2019) Long-term after treatment with platinum compounds, cisplatin and [Pt(O,O'-acac )(γ-acac)(DMS)]: autophagy activation in rat B50 neuroblastoma cells. Toxicol Appl Pharmacol 364:1–11CrossRefGoogle Scholar
  16. Gump JM, Thorburn A (2011) Autophagy and apoptosis: what is the connection? Trends Cell Biol 21:387–392CrossRefGoogle Scholar
  17. Han Y, Fan S, Qin T, Yang J, Sun Y, Lu Y, Mao J, Li L (2018) Role of autophagy in breast cancer and breast cancer stem cells (review). Int J Oncol 52:1057–1070Google Scholar
  18. Hurley JH, Young LN (2017) Mechanisms of autophagy initiation. Annu Rev Biochem 86:225–244CrossRefGoogle Scholar
  19. Kaushal V, Herzog C, Haun RS, Kaushal GP (2014) Caspase protocols in mice. Methods Mol Biol 1133:141–154CrossRefGoogle Scholar
  20. Kishi-Itakura C, Koyama-Honda I, Itakura E, Mizushima N (2014) Ultrastructural analysis of autophagosome organization using mammalian autophagy-deficient cells. J Cell Sci 127:4089–4102CrossRefGoogle Scholar
  21. Koppel H, Lewis PD, Padel AJ (1983) Cell death in the external granular layer of normal and undernourished rats: futher observations, including estimates of rate of cell loss. Cell Tissue Kinet 16:99–106Google Scholar
  22. Lebwohl M, Menter A, Koo J, Feldman SR (2004) Combination therapy to treat moderate to severe psoriasis. J Am Acad Dermatol 50:416–430CrossRefGoogle Scholar
  23. Levine B, Packer M, Codogno P (2015) Development of autophagy inducers in clinical medicine. J Clin Invest 125:14–24CrossRefGoogle Scholar
  24. Li L, Wang Y, Jiao L, Lin C, Lu C, Zhang K, Hu C, Ye J, Zhang D, Wu H, Feng M, He Y (2019) Protective autophagy decreases osimertinib cytotoxicity through regulation of stem cell-like properties in lung cancer. Cancer Lett 452:191–202CrossRefGoogle Scholar
  25. Lossi L, Merighi A (2003) In vivo cellular and molecular mechanisms of neuronal apoptosis in the mammalian CNS. Prog Neurobiol 69:287–312CrossRefGoogle Scholar
  26. Lumkwana D, du Toit A, Kinnear C, Loos B (2017) Autophagic flux control in neurodegeneration: progress and precision targeting - where do we stand? Prog Neurobiol 153:64–85CrossRefGoogle Scholar
  27. Manto M (2012) Toxic agents causing cerebellar ataxias. Handb Clin Neurol 103:201–213CrossRefGoogle Scholar
  28. Martí J, Santa-Cruz MC, Serra R, Hervás JP (2015) Systematic differences in time of cerebellar-neuron origin derived from bromodeoxyuridine immunoperoxidase staining protocols and tritiated thymidine autoradiographic: a comparative study. Int J Dev Neurosci 47:216–228CrossRefGoogle Scholar
  29. Martí J, Santa-Cruz MC, Serra R, Hervás JP (2016) Hydroxyurea treatment and development of the rat cerebellum: effects on the neurogenetic profiles and settled patterns of Purkinje cells and deep cerebellar nuclei neurons. Neurotox Res 30:563–580CrossRefGoogle Scholar
  30. Martí J, Molina V, Santa-Cruz MC, Hervás JP (2017) Developmental injury to the cerebellar cortex following hydroxyurea treatment in early postnatal life: an immunohistochemical and electron microscopic study. Neurotox Res 31:187–203CrossRefGoogle Scholar
  31. McGann PT, Ware RE (2015) Hydroxyurea therapy for sickle cell anemia. Expert Opin Drug Saf 14:1749–1758CrossRefGoogle Scholar
  32. Navarra P, Preziosi P (1999) Hydroxyurea: new insights on an old drug. Crit Rev Oncol Hematol 29:249–255CrossRefGoogle Scholar
  33. Nazha A, Khoury JD, Verstovsek S, Daver N (2016) Second line therapies in polycythemia vera: what is the optimal strategy after hydroxyurea failure? Crit Rev Oncol Hematol 105:112–117CrossRefGoogle Scholar
  34. Newton HB (2007) Hydroxyurea chemotherapy in the treatment of meningiomas. Neurosurg Focus 23(4):E11CrossRefGoogle Scholar
  35. Patxinos G, Watson C (1998) The rat brain in stereotaxic coordinates. Fourth edition. Academic Press, San DiegoGoogle Scholar
  36. Rodríguez-Vázquez L, Martí J (2017) Effects of hydroxyurea exposure on the rat cerebellar neuroepithelium: an immunohistochemical and electron microscopic study along the anteroposterior and mediolateral axes. Neurotox Res 32:671–682CrossRefGoogle Scholar
  37. Rodríguez-Vázquez L, Vons O, Valero O, Martí J (2019) Hydroxyurea exposure and development of the cerebellar external granular layer: effects on granule cell precursors, Bergmann glial and microglial cells. Neurotox Res 35:387–400CrossRefGoogle Scholar
  38. Roux C, Aligny C, Lesueur C, Girault V, Brunel V, Ramdani Y, Genty D, Driouich A, Laquerrière A, Marret S, Brasse-Lagnel C, Gonzalez BJ, Bekri S (2015) NMDA receptor blockade in the developing cortex induces autophagy-mediated death of immature cortical GABAergic interneurons: an ex vivo and in vivo study in Gad67-GFP mice. Exp Neurol 267:177–193CrossRefGoogle Scholar
  39. Saban N, Bujak M (2009) Hydroxyurea and hydroxamic acid derivatives as antitumor drugs. Cancer Chemother Pharmacol 64:213–221CrossRefGoogle Scholar
  40. Sampson M, Archibong AE, Powell A, Strange B, Roberson S, Hills ER, Bourne P (2010) Perturbation of the developmental potential of preimplantation mouse embryos by hydroxyurea. Int J Environ Res Public Health 7:2033–2044CrossRefGoogle Scholar
  41. Schlisser AE, Hales BF (2013) Deprenyl enhances the teratogenicity of hydroxyurea in organogenesis stage mouse embryos. Toxicol Sci 134:391–399CrossRefGoogle Scholar
  42. Shao J, Zhou B, Chu B, Yen Y (2006) Ribonucleotide reductase inhibitors and future drug design. Curr Cancer Drug Targets 6:409–431CrossRefGoogle Scholar
  43. Song S, Tan J, Miao Y, Li M, Zhang Q (2017) Crosstalk of autophagy and apoptosis: involvement of the dual role of autophagy under ER stress. J Cell pçhysiol 232:2977–2984CrossRefGoogle Scholar
  44. Sotelo C (2015) Molecular layer interneurons of the cerebellum: developmental and morphological aspects. Cerebellum 14:534–556CrossRefGoogle Scholar
  45. Taylor MA, Das BC, Ray SK (2018) Targeting autophagy for combating chemoresistance and radioresistance in glioblastoma. Apoptosis 23:563–575CrossRefGoogle Scholar
  46. Wang Y, Zhou K, Li T, Xu Y, Xie C, Sun Y, Zhang Y, Rodríguez J, Blomgren K, Zhu C (2017) Inhibition of autophagy prevents irradiation-induced neural stem and progenitor cell death in the juvenile mouse brain. Cell Death Dis 23:e2694CrossRefGoogle Scholar
  47. Woo GH, Katayama K, Jung JY, Uetsuka K, Bak EJ, Nakayama H, Doi K (2003) Hydroxyurea (HU)-induced apoptosis in the mouse fetal tissues. Histol Histopathol 18:387–392Google Scholar
  48. Woo GH, Katayama K, Bak EJ, Ueno H, Tamauchi H, Uetsuka K, Nakayama H, Doi K (2004) Effects of prenatal hydroxyurea-treatment on mouse offspring. Exp Toxicol Pathol 56(1–2):1–7CrossRefGoogle Scholar
  49. Woo GH, Bak EJ, Katayama K, Doi K (2006) Molecular mechanisms of hydroxyurea (HU)-induced apoptosis in the mouse fetal brain. Neurotocol Teratol 28:125–134CrossRefGoogle Scholar
  50. Zala C, Rouleau D, Montaner JS (2000) Role of hydroxyurea in treatment of disease due to human immunodeficiency virus infection. Clin Infect Dis 2000(Suppl 2):S143–S150CrossRefGoogle Scholar
  51. Zhang L, Goldman JE (1996) Generation of cerebellar interneurons from dividing progenitors in white matter. Neuron 16:(1)47–54CrossRefGoogle Scholar
  52. Zhang XY, Zhang M, Cong Q, Zhang MX, Zhang MY, Lu YY, Xu CJ (2018) Hexokinase 2 confers resistance to cisplatin in ovarian cancer cells by enhancing cisplatin-induced autophagy. Int J Biochem Cell Biol 95:9–16CrossRefGoogle Scholar
  53. Zhi X, Feng W, Rong Y, Liu R (2018) Anatomy of autophagy: from the beginning to the end. Cell Mol Life Sci 75:815–831CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Unidad de Citología e Histología, Facultad de BiocienciasUniversidad Autónoma de BarcelonaBellaterraSpain

Personalised recommendations