frontal cortex, neurons, antenatal alcoholization.The aim of the present work was to undertake a comparative study of the effects of prenatal alcoholization of the histological characteristics of neurons in the frontal cortex of the brains of rats of different ages. Experiments were performed on 175 mongrel white rats - the offspring of 25 females given 15% ethanol as drinking solution throughout pregnancy. The cerebral cortex was studied on postnatal days 2-90 using histological, histochemical, and morphometric methods. An increase (on days 2 and 5) followed by a decrease (on days 10 and 90) was seen in cortical thickness, with a decrease in neuron body size (on days 20-90), a decrease in the number of neurons in cortical layer V, a decrease in the number of normochromic cells, and increases in the numbers of hyperchromic shrunken neurons and ghost cells throughout the study period. Antenatal alcoholization in rats induced various histological changes in the frontal cortex, which were long-lasting and progressive during postnatal ontogeny

Bon, L. I.

Zimatkin, S. M.

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3700097-0549/17/4703-0370 ©2017 Springer Science+Business Media New YorkNeuroscience and Behavioral Physiology, Vol. 47, No. 3, March, 2017 Alcohol consumption during pregnancy leads to the de-velopment of a number of specifi c injuries to the fetal body, which are combined in the concept of fetal alcohol syndrome, which is among the fetal alcohol spectrum disorders (FASD) [2, 14]. Published data indicate that the cerebral cortex is par-ticularly sensitive to prenatal alcohol [4, 5], which in rodents induces neuron apoptosis and neurodegenerative changes [4]. Prenatal alcohol decreases the numbers and sizes of pyrami-dal neurons in the cerebral cortex in animals, decreasing their protein content and producing underdevelopment of the cyto-plasm [7]. The sensorimotor cortex in rat pups shows signs of delayed development of neurons and their dendrites, with destructive and dystrophic changes to cells (karyocytolysis, chromatolysis, appearance of “ghost cells”), with signifi cant ultrastructural damage [4, 14]. However, the dynamics of his-tological changes during postnatal ontogeny in these animals has received insuffi cient study. The aim of the present work was to undertake a com-parative study of the effects of prenatal alcoholization on the histological characteristics of neurons in the frontal cor-tex in rats of different ages. Materials and Methods Experiments were performed on 25 female mongrel white rats with a starting weight of 230 ± 20 g and their off-spring (175 rat pups). All experiments were performed in compliance with the “Regulations for Studies Using Experi-mental Animals” [6]. The study was approved by the Biome-dical Ethics Committee (Protocol No. 7 of December 23, 2013) of Grodno State Medical University. Animals were kept on a standard animal house diet. Rats of the experimen-tal group received 15% ethanol solution as the sole drinking fl uid throughout pregnancy (from the day on which spermato-zoids were detected in vaginal smears until parturition), while control animals received the same quantity of water. Mean alcohol consumption by pregnant females was 4 ± 2 g/kg/day. On days 2, 5, 10, 20, 45, and 90 after birth, rat pups were weighed and decapitated, and brain weight was determined, along with the brain:body weight ratio. Fragments of the an-terior part of the cerebral cortex were rapidly fi xed in Carnoy’s solution. Serial paraffi n sections were stained with 0.1% toluidine blue by the Nissl method; ribonucleoproteins (RNP) were detected using the Einarsson method.Dynamics of Histological Changes in the Frontal Cortex of the Brain in Rats Subjected to Antenatal Exposure to AlcoholS. M. Zimatkin and E. I. Bon’ UDC 611.813.1-018:613.81:599.323.4Translated from Morfologiya, Vol. 149, No. 2, pp. 11–15, March–April, 2016. Original article submitted April 13, 2016. Revised version received November 28, 2016.The aim of the present work was to undertake a comparative study of the effects of prenatal alcoholization of the histological characteristics of neurons in the frontal cortex of the brains of rats of different ages. Experiments were performed on 175 mongrel white rats – the offspring of 25 females given 15% ethanol as drinking solution throughout pregnancy. The cerebral cortex was studied on postnatal days 2–90 using his-tological, histochemical, and morphometric methods. An increase (on days 2 and 5) followed by a decrease (on days 10 and 90) was seen in cortical thickness, with a decrease in neuron body size (on days 20–90), a decrease in the number of neurons in cortical layer V, a decrease in the number of normochromic cells, and increases in the numbers of hyperchromic shrunken neurons and ghost cells throughout the study peri-od. Antenatal alcoholization in rats induced various histological changes in the frontal cortex, which were long-lasting and progressive during postnatal ontogeny.Keywords: frontal cortex, neurons, antenatal alcoholization.Department of Histology, Cytology, and Embryology, Grodno State Medical University, Grodno, Belarus; e-mail: zimatkin@grsmu.by.DOI 10.1007/s11055-017-0407-1371Dynamics of Histological Changes in the Frontal Cortex of the Brain Histological studies, microphotography, morphometry, and densitometry of chromogen deposits in histological preparations were performed using an Axioscop 2 plus mi-croscope (Zeiss, Germany) with a Leica DFC 320 digital video camera (Leica, Germany) and the ImageWarp image analysis program (Bitfl ow, USA). The position of the fron-tal cortex on histological sections of the rat brain was iden-tifi ed using a stereotaxic atlas [13]. Preparations stained by the Nissl method were used to measure the thickness of the frontal cortex (fi ve measurements in each animal) and neu-ron density was measured in layer V (fi ve microscope fi elds for each animal), and among these the numbers of cells with different levels of cytoplasmic chromatophilia (hypo-, hy-perchromic, hyperchromic shrunken, and ghost cells) were counted. The small and large diameters of neuron bodies were measured, and the cross-sectional areas, elongation factors, and form factors were calculated. At least 30 neu-rons from each animal and at least 150 from each group were examined, ensuring a suffi ciently large sample set for subsequent analysis. Mean values determined for numerical data from each animal were analyzed using nonparametric statistics in Statistica 6.0 for Windows (StatSoft Inc., USA). Descriptive statistics for each parameter were the median (Me), percen-tile boundaries (from 25 to 75), and the interquartile range (IQR). Numerical results are presented as Me (median), LQ (upper boundary of lower quartile), and UQ (lower bound-ary of upper quartile). Differences between parameters in the control and experimental group were regarded as signif-icant at p < 0.05 (Mann and Whitney U test) [1]. Results There were no signifi cant differences in body weight, brain weight, or the brain:body weight ratio between control and experimental rat pups. Control pups displayed thicken-ing of the frontal cortex throughout the observation period, while the frontal cortex of pups exposed to antenatal alco-holization was signifi cantly thicker, by 33% on postnatal day 2 and by 13% on day 5, than in controls, while thickness on day 10 showed a statistically signifi cant (by 12%) de-crease. These differences were no longer present on days 20 or 45, though on day 90 there was a further decrease in cor-tex thickness, by 23%, compared with controls (Table 1). Cortical layer V of alcoholized rat pups showed a sig-nifi cant decrease (by 10–25%) in the number of neurons per unit section area (density) at all time points studied (see Table 1). In control rats, Nissl-stained preparations showed that the ratio of neurons with different levels of cytoplasmic chro-matophilia differed signifi cantly at different times of postna-tal development; however, normochromic neurons were al-ways dominant (Fig. 1, a) (the proportion was 60–70% of all neurons). In the experimental animals, layer V of the cerebral cor-tex showed a decreased number of normochromic neurons and increased numbers of hyperchromic shrunken neurons and ghost cells at all time points, the intensity of these chang-es differing at different periods of postnatal development (see Fig. 1, b). The smallest changes were seen on day 2, where the increases in the numbers of hyper- and hypochro-mic neurons were statistically insignifi cant. The largest TABLE 1. Characteristics of the Frontal Cortex in Rat Pups in Controls and after Prenatal Alcoholization, Me (LQ; UQ).Study parameterGroup of rat pupsTime point, days2 5 10 20 45 90Cortical thickness, μmControl538 (532;567)695 (679;711)1156 (1131;1182)1277 (1251;1307)1465 (1444;1473)1645 (1584;1654)Experimental809* (802;823)799* (796;880)1020* (1016;1024)1257 (1256;1262)1445 (1379;1488)1249* (1225;1262)Neuron density, number per 1 mm2 of section areaControl16819 (16684;17223)9553 (9553;9688)7804 (7669;7804)6122 (6055;6189)5180 (4978;5247)4575 (4306;4575)Experimental13993* (13724;14263)8880* (8880;9015)6122* (6055;6728)5382* (5247;5382)3767* (3767;3902)3229* (3095;3229)Hyperchromic shrunken neurons, number per 1 mm2 of section areaControl0 (0;0)0 (0;0)0 (0;0)134 (134;134)134 (134;134)0 (0;0)Experimental0 (0;0)0 (0;0)0 (0;0)336* (269;404)404* (404;538)404* (404;404)Layer V neuron ribonucleoprotein content, OD unitsControl0.23 (0.22;0.23)0.20 (0.18;0.22)0.20 (0.18;0.22)0.14 (0.13;0.15)0.19 (0.18;0.2)0.125 (0.12;0.13)Experimental0.23 (0.2;0.24)0.26* (0.2;0.28)0.22 (0.19;0.26)0.16 (0.145;0.165)0.20 (0.19;0.21)0.18* (0.17;0.19)*Signifi cant difference compared with control, p < 0.05.  Me – median; numbers in parentheses are quartile boundaries (from 25 to 75).372 Zimatkin and Bon’changes in the frontal cortex were seen on days 20–90 of postnatal development. Thus, the number of hyperchromic shrunken neurons was increased by 66% on day 45, while the numbers of hypochromic neurons and ghost cells in-creased by 20% and 40%, respectively, compared with con-trols (Fig. 2). Hyperchromic shrunken neurons in control animals were almost absent on days 45 and 90, while the numbers of these cells, conversely, increased sharply in ani-mals subjected to antenatal alcoholization (Fig. 3). Studies of the sizes and shapes of neuron bodies re-vealed a statistically signifi cant transient increase in the cross-sectional area of neuron perikarya in layer V on day 2. However, on days 20–90 of postnatal development, neuron body size in experimental rat pups became signifi cantly smaller than that in controls (see Fig. 3), while neuron size progressively increased in control animals. In rats subjected to antenatal alcoholization, the growth of neuron bodies ceased after 10–20 days of postnatal development (see Fig. 3). There was a negative correlation between neuron cross-sec-tional area and the number of hyperchromic shrunken neu-rons (r = –(0.87–0.98); p < 0.05). The RNP content in the cytoplasm of frontal cortex layer V neurons in alcoholized rats was signifi cantly greater on days 5 and 90 (see Table 1) than in controls, which cor-related with increases in the number of hyperchromic neu-rons at these time points (r = 0.86–0.96, p < 0.05). Discussion The thickening of the cortex seen here in antenatal al-coholized rat pups on postnatal day 5 and especially day 2 may be linked with the perivascular edema and neuron swelling seen on histological preparations. The strongest correlation between cortical thickness and neuron body cross-sectional area (r = 0.98, p < 0.01) was seen on day 2. The subsequent decrease in cortical thickness on day 10 Fig. 1. Neurons in frontal cortex layer V in 45-day-old rat pups of the control group (a) and after antenatal alcoholization (b). a) Dominance of normo-chromic neurons; b) dominance of hyperchromic and hyperchromic shrunken neurons. Stained with thionine, Nissl method. Magnifi cation ×400Fig. 2. Ratios of types of neuron with different chromatophilias in the frontal cortex in 45-day-old rat pups: control (a) and after antenatal alcoholization (b).373Dynamics of Histological Changes in the Frontal Cortex of the Brainmay be associated with the disappearance of edema, while recurrent thinning on day 90 may be due to total shrinkage of neurons, which is supported by the strong correlation be-tween cortical thickness and the number of shrunken neu-rons (r = –0.94, p < 0.01). At the same time, cortical thick-ness in control animals showed a consistent growth. Size reductions and shrinkage of neurons have been reported previously in adult rats subjected to antenatal alco-holization [7, 9, 10]. In both study groups, postnatal ontogeny was associat-ed with a decrease in the density of neuron bodies, which may be linked with more intense growth of the neuropil as compared with neuron bodies. The decrease in neuron den-sity seen here in the frontal cortex of experimental rats at all postnatal time points may be associated with the death of a proportion of neurons due to alcohol exposure during em-bryogenesis. This neuron defi cit persists lifelong. Decreases in cortical thickness and the numbers of neurons within it and increases in the numbers of hyperchromic shrunken neurons and ghost cells at different postnatal time points in the offspring of rats consuming alcohol during pregnancy have been reported by other authors [12]. The cessation of growth and the shrinkage of neurons seen in antenatally alcoholized rats from postnatal day 20 may be linked with impairment to water-salt metabolism and the cell cytoskeleton, as well as with oxidative stress, activation of lipid peroxidation, and oxidation of proteins. Free radicals interact with DNA and produce structural modifi cations. In addition, free radicals damage cell mem-branes and also the membranes of organelles in neurons, particularly mitochondria, while the level of the endoge-nous antioxidant glutathione decreases [9]. Alcohol impairs transcription and translation processes in the developing brain and induces derangements of gene expression [11]. Electron microscopic studies have demonstrated increased cytoplasmic density in hyperchromic neurons, with cleft-like lightened areas and damaged organelles, deep invagina-tions into the nuclear envelope, breakup of Golgi complex cisterns into vacuoles, and severe swelling of mitochondria [7]. Published data also indicate that ethanol inhibits the ex-pression of endogenous insulin and insulin-like growth fac-tor (IGF) polypeptides, as well as IGF1 and IGF2 receptors in the brain. The results demonstrate that the main impair-ments in the brain in FASD are due to deranged insulin/IGF signaling [10]. These structural changes may underlie the known neuro-logical and behavioral impairments in animals after antenatal alcoholization. Behavioral impairments include cognitive [3], sensorimotor, and emotional disorders [4], Neuro logical dys-functions evoked by antenatal alcoholization include audito-ry dysfunction, speech delay, and inability to communicate and learn [3]. In particular, antenatal alcoholization leads to decreased motor activity in the open fi eld test in rat pups at age 17 days and has negative effects on their ability to acquire (but not to reproduce) a conditioned food-related refl ex. In addition, animals develop tolerance to alcohol [8]. Thus, antenatal alcoholization produces profound and varied histological changes in the frontal cortex in rats, which during postnatal ontogeny are wavelike, long-lasting, and sometimes progressive. Thus, there was an increase (days 2 and 5) and then a decrease (days 10 and 90) in cor-tical thickness and neuron size (days 20–90), a decrease in the number of neurons in cortical layer V, and decreases in the numbers of hyperchromic shrunken neurons and ghost cells at all time points. Of particular interest are the cessa-tion of growth and the progressive shrinkage of neurons in the frontal cortex from day 20 of postnatal development.REFERENCES  1. N. V. Batin, Computerized Statistical Data Analysis: Textbook, Institute of Scientifi c Training, Belarus Academy of Sciences, Minsk (2008).  2. S. M. Zimatkin and E. I. Bon’, “Antenatal alcoholization: damage to the internal organs,” Nov. Med. Biol. Nauk., No. 2, 168–174 (2013).  3. S. M. Zimatkin and E. I. Bon’, “Fetal alcohol syndrome: behavioral and neurological impairments,” Zh. Grodn. Med. Univ., No. 2, 14–17 (2013).  4. S. M. Zimatkin and E. I. Bon’, Fetal Alcohol Syndrome: Monograph, Novoe Znanie, Minsk (2014).  5. S. M. Zimatkin and E. I. Bon’, “Effects of alcohol on the developing brain,” Morfologiya, 145, No. 2, 79–88 (2014).  6. N. N. Karkishchenko and S. V. Gracheva, Handbook for Laboratory Animals and Alternative Models in Biomedical Studies, Profi l-2S, Moscow (2010).  7. E. N. Popova, Ultrastructure of the Brain, Alcohol, and Offspring, Nauchnyi Mir, Moscow (2010).  8. E. L. Abel, “In utero alcohol exposure and development delay of response inhibition.” Alcoholism, 6, 369–376 (1982).  9. P. S. Brocardo, “Anxiety- and depression-like behaviors are accom-panied by an increase in oxidative stress in a rat model of fetal alco-hol spectrum disorders: Protective effects of voluntary physical ex-ercise,” Neuropharmacology, 62, 16–18 (2012).10. S. M. de la Monte and J. R. Wands, “Role of central nervous system insulin resistance in fetal alcohol spectrum disorders,” J. Popul. Ther. Clin. Pharmacol., 17, No. 3, 390–404 (2010).Fig. 3. Cross-sectional areas of frontal cortex layer V neuron bodies in control rat pups (I) and rat pups subjected to antenatal alcoholization (II). The abscissa shows age (days) and the ordinate shows study parameters (μm2). *Signifi cant differences compared with controls, p < 0.05. Vertical bars show interquartile ranges.374 Zimatkin and Bon’11. M. L. Kleiber and E. Wright, “Maternal voluntary drinking in C57BL/ 6J mice: advancing a model for fetal alcohol spectrum disorders,” Behav. Brain Res., 223, 376–387 (2011).12. D. Lopez-Tejero, “Effects of prenatal ethanol exposure on physical growth sensory refl ex maturation and brain development in the rat,” Neuropathol. Appl. Neurobiol., 12, No. 3, 251–260 (1986).13. G. Paxinos and C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, London (1998).14. E. P. Riley, M. A. Infante, and K. R. Warren, “Fetal alcohol spectrum disorders: an overview,” Neuropsychol. Rev., 21, 73–80 (2011).

Dynamics of Histological Changes in the Frontal Cortex of the Brain in Rats Subjected to Antenatal Exposure to Alcohol

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Dynamics of Histological Changes in the Frontal Cortex of the Brain in Rats Subjected to Antenatal Exposure to Alcohol

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