Contents lists available at ScienceDirect Journal of Chemical Neuroanatomy journal homepage: www.elsevier.com/locate/jchemneu The effects of maternal diabetes and insulin treatment on neurogenesis in the developing hippocampus of male rats Akram Sadeghi a,b , Ebrahim Esfandiary c , Javad Hami a,b , Hossein Khanahmad d , Zahra Hejazi d , Mohammad Mardani c , Shahnaz Razavi c, ⁎ a Cellular and molecular Research Center, Birjand University of Medical Sciences, Birjand, Iran b Department of Anatomical Sciences, School of Medicine, Birjand University of Medical Sciences, Birjand, Iran c Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran d Department of Genetic and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran ARTICLE INFO Keywords: Maternal diabetes Neurogenesis Hippocampus Rat neonates ABSTRACT Diabetes in pregnancy is associated with an increasing risk of congenital malformations and central nervous system disorders (CNS) especially hippocampal neuronal circuitry disruption as a discreet region involved in neurogenesis phenomenon. This study aimed to investigate the effect of maternal diabetes and insulin treatment on the expression and distribution pattern of NeuN and DCX as two important markers of neurogenesis paradigm in developing rat hippocampus. All animals were randomly divided into three groups as follows: Control group, Diabetic (STZ-D), Diabetic treated with insulin (STZ-INS). Diabetes was induced in Wistar female rats by Sterptozotocin intraperitoneal injection (single does). Following confirmation of diabetes, animals were mated with non-diabetic males. Four to six units of protamine-Zinc insulin were delivered subcutaneously (SC) in insulin treated group. At the post-natal day 14 (P14), the brain of male offspring’s were removed for further study. In fact Immunofluorescence staining and Real time - PCR assays are used for evaluation of neurogenesis phenomenon. Our results showed a significant higher level of hippocampal DCX expression and an increase in the mean number of DCX positive cells in the DG of diabetic group male offspring (P < 0.05). We also found an insignificant up-regulation in the expression of DCX and the mean number of positive cells in the insulin-treated diabetic group neonates as compared to control group (P > 0.05). Nevertheless the results of immuno- fluorescence staining for NeuN also indicated that the mean number of NeuN+ cells was significantly lower in dentate gyrus of diabetic group male offspring (P < 0.05). Besides, there were significant down- regulation in the hippocampal mRNA expression of NeuN in diabetic pups compare to control (P < 0.05 each). Our results revealed that diabetes during pregnancy has an adverse effect on the hippocampal neurogenesis in rat neonates. Furthermore, the control of glycemia by insulin is sufficient to prevent the alterations in expression of neuro- genesis markers. 1. Introduction 1.1. Diabetes mellitus and CNS development Diabetes mellitus is one of the most common disorders in pregnancy that is thought to affect up to 15 percent of pregnancies worldwide (Chiu and Cline, 2010; Hallschmid et al., 2007; Needleman and McAllister, 2008). This serious metabolic condition is associated with an increased risk of mortality and morbidity as well as congenital malformation in multiple vital systems such as urinary, respiratory and central nervous systems (CNS) in the offspring (Churchill et al., 1969; Georgieff, 2006; Schwartz and Teramo, 2000). The risk of the devel- oping fetal abnormalities is due in part to increased placental transport of glucose and other nutrients from the mother to the fetus. Therefore, maternal hyperglycemia is paralleled by fetal hyperglycemia that sti- mulates the pancreatic β-cell hypertrophy and hyperplasia in devel- oping fetus resulting in increased insulin secretion and consequently, in utero hyperinsulinemia which affects the developing organ systems at different developmental times (Pedersen et al., 1981; Salvesen et al., 1993; Susa et al., 1984; Widness et al., 1981). The multiple lines of https://doi.org/10.1016/j.jchemneu.2018.03.005 Received 30 January 2018; Received in revised form 17 March 2018; Accepted 21 March 2018 ⁎ Corresponding author. E-mail addresses: akramsadeghi944@gmail.com (A. Sadeghi), razav@medmui.ac.ir (S. Razavi). Abbreviations: STZ, Stereptomycin; DCX, doublecrtin; NeuN, Neuronal nuclear antigen; DG, dentate Gyrus; ROS, reactive oxygen species; IGF-1, Insulin like growth factor 1; CNS, Central nervous system Journal of Chemical Neuroanatomy 91 (2018) 27–34 Available online 22 March 2018 0891-0618/ © 2018 Elsevier B.V. All rights reserved. T