ORIGINAL ARTICLE Patterns of Apoptosis and Autophagy Activation After Hydroxyurea Exposure in the Rat Cerebellar External Granular Layer: an Immunoperoxidase and Ultrastructural Analysis Vanessa Molina 1 & Lucía Rodríguez-Vázquez 1 & Joaquín Martí 1 Received: 23 May 2019 /Revised: 24 July 2019 /Accepted: 30 July 2019 # Springer Science+Business Media, LLC, part of Springer Nature 2019 Abstract 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. Keywords Perinatal . Cerebellar cortex . External granular layer . Hydroxyurea . Apoptosis . Autophagy . Electron microscopy Introduction Hydroxyurea (HU), a hydroxylated analog of urea, is a water- soluble, non-alkylating compound used to increase fetal he- moglobin concentration in patients with sickle cell anemia (McGann and Ware 2015). It is also used in the treatment of a wide range of diseases, including psoriasis (Lebwohl et al. 2004), HIV infection (Zala et al. 2000), myeloproliferative disorders (Navarra and Preziosi 1999), and carcinomas (Saban and Bujak 2009). HU suppresses DNA replication via inhibition of the class I ribonucleotide reductase by inactivating the tyrosyl radical required for enzyme activity (Shao et al. 2006; Saban and Bujak 2009). The use of HU is based on its ability to arrest proliferating cell populations in the G1/S phase of the cell cycle (Doi 2011). When tested in laboratory animals, the administration of HU to pregnant rodents compromises the ability of embryos to develop (Sampson et al. 2010) and induces alterations in their craniofacial tissues (Schlisser and Hales 2013). In addi- tion, growth retardation (Woo et al. 2004) and apoptosis of neuroepithelial cells in the fetal telencephalon (Woo et al. 2003, 2006) have also been reported. Data from our laboratory have revealed that, in the embryonic life, this agent induces apoptosis in the cerebellar neuroepithelium (Rodríguez- Vázquez and Martí 2017), depletes the number of Purkinje cells and deep cerebellar nuclei neurons, and alters the neurogenetic profiles of these macroneurons (Martí et al. 2016). In the postnatal life, on the other hand, HU induces a myriad of effects such as apoptosis in the cerebellar external granular layer (EGL), overexpression of the cytoskeletal pro- tein vimentin, alterations in the cytoarchitectonics of the cer- ebellar cortex, and activation of the microglial cells (Martí et al. 2017; Rodríguez-Vázquez et al. 2019). Autophagy, also known as type 2 cell death (Lossi and Merighi 2003), is an evolutionary conserved intracellular pro- cess through which proteins and cellular components are se- questered in vesicles called autophagosomes and degraded upon fusion with lysosomal compartments (Zhi et al. 2018; * Joaquín Martí joaquim.marti.clua@uab.es 1 Unidad de Citología e Histología, Facultad de Biociencias, Universidad Autónoma de Barcelona, 08193 Bellaterra, Barcelona, Spain Neurotoxicity Research https://doi.org/10.1007/s12640-019-00094-y