Volumetric investigation of the hippocampus in rat offspring due to diabetes in pregnancy–A stereological study

Journal Pre-proof Volumetric Investigation of the Hippocampus in Rat Offspring Due to Diabetes in Pregnancy- A Stereological Study Akram Sadeghi, Hadi Asghari, Javad Hami, Mina Mohasel Roodi, Hamideh Mostafaee, Mohammad Karimipour, Mohamadreza Namavar, Faezeh Idoon

PII:

S0891-0618(18)30204-7

DOI:

https://doi.org/10.1016/j.jchemneu.2019.101669

Article Number:

101669

Reference:

CHENEU 101669

To appear in: Received Date:

6 December 2018

Revised Date:

23 June 2019

Accepted Date:

19 August 2019

Please cite this article as: Sadeghi A, Asghari H, Hami J, Mohasel Roodi M, Mostafaee H, Karimipour M, Namavar M, Idoon F, Volumetric Investigation of the Hippocampus in Rat Offspring Due to Diabetes in Pregnancy- A Stereological Study, Journal of Chemical Neuroanatomy (2019), doi: https://doi.org/10.1016/j.jchemneu.2019.101669

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Volumetric Investigation of the Hippocampus in Rat Offspring Due to Diabetes in Pregnancy- A Stereological Study Running Title: Stereological evaluation of the newborns' hippocampus

Authors: Akram Sadeghi 1,2, Hadi Asghari 3, Javad Hami 1,2, Mina Mohasel roodi 2, Hamideh Mostafaee 2, Mohammad karimipour 4, Mohamadreza Namavar5, Faezeh Idoon 2. Affiliations: Cardiovascular Diseases Research Center, Birjand University of Medical Sciences, Birjand, Iran.

2.

Department of Anatomy and Cell Biology, School of Medicine, Birjand University of Medical Sciences, Birjand, Iran.

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Institute of Microstructure Technology (ITM), Karlsruhe Institute of Technology Karlsruhe, Germany

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Department of Anatomical Sciences, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.

5.

Clinical Neurology Research Center, and Histomorphometry & Stereology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.

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Highlights

The brain development during the prenatal period is affected by various factors including

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mother's metabolic condition. we hypothesize that the learning and memory impairment observed

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in the offspring of diabetic mothers might be related to a loss of neuronal density and volume in their hippocampus. In this study, by using cresyl violet staining and stereological methods, we investigate the volume and the numerical density of the hippocampal neurons in the two-week old rats born to diabetic dams and compare with the normal animals. Bilateral hippocampal volume

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decreased in the diabetic group, mainly in the CA1, dentate gyrus (DG) and subiculum areas

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Abstract Background: The brain development during the prenatal period is affected by various factors, including the mother's metabolic condition. It has been revealed that diabetes in pregnancy is associated with structural and functional alterations in offspring’s hippocampus. Hippocampus, as a critical region with well-known roles in learning and memory consolidation, is vulnerable to changes in glucose level. This study was designed to investigate the effects of maternal diabetes

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during the pregnancy period and insulin therapy on the neuronal density and the volume of

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different subfields of the hippocampus in rat offspring at postnatal day 14 (P14).

Methods: Wistar female rats were randomly divided into diabetics (STZ-D), diabetes treated

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with insulin (STZ-INS) group, and controls (CON). The animals in all groups were mated by

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non-diabetic male rats. Two weeks after birth, male pups from each group were sacrificed. The Cavalieri method was carried out to estimate the total volume, and the numerical density of the

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neurons in the hippocampus and its sub regions was measured by the optical dissector technique. Results: Bilateral hippocampal volume decreased in the diabetic group, mainly in the CA1,

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dentate gyrus (DG) and subiculum areas (P≤0.05), when compared to control and insulin-treated diabetic animals. In all hippocampus sub-regions, maternal diabetes resulted in a significant decrease in the number of cells in comparison with two other groups (P≤0.01 each). Conclusion: These data indicate that diabetes during pregnancy has a negative impact on the

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development of the hippocampus in the rats. These changes in the volume of hippocampal CA1, DG, and subiculum areas might be at the core of underlying neurocognitive and neurobehavioral impairments observed in the children of diabetic mothers. Key word: Maternal Diabetes; Hippocampus, Neuronal density; Volume; Stereology.

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Introduction: Diabetes Mellitus (DM) during the pregnancy period is a very serious metabolic condition that affects up to 15 percent of pregnancies throughout the globe (1-3). This metabolic condition is associated with an increased risk of maternal and child mortality and morbidity (4, 5). Multiple lines of studies found that maternal diabetes has a negative impact on the development of the central nervous system (CNS), characterized by neuro-cognitive and neuro-behavioral deficits

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observed in their offspring (6-9).

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The previous animal and human studies have shown that infants born to diabetic mothers are at risk of having neurodevelopmental sequelae and neurocognitive disorders (10, 11), Including

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cerebral dysfunction (12), neurological abnormalities (13), learning and memory impairments

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(14-17). There is striking evidence demonstrating that the offspring of diabetic mothers have lower Intelligence Quotient (IQ) scores and general cognitive functions than normal children (14,

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18, 19). Interestingly, there are reports illustrating a close relationship between maternal diabetes in pregnancy and an increased risk of psychological disorders such as schizophrenia in the

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offspring (12, 20-24).

The hippocampus - an important part of the limbic system - plays a critical role in learning, memory formation, and spatial navigation (25, 26). This part of the brain is particularly vulnerable to the changes in glucose concentration, (i.e., hyper – and hypoglycemia), particularly

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during the CNS development (27-29). Several studies have reported that hyperglycemia affects the proliferation, migration, differentiation, and survival of hippocampal cells (30-33). On the other hand, experimental studies have indicated a decrease in the numerical density of neurons in the cerebrum and cerebellum of offspring born to diabetic animals (34-37). In a recent study by Chandna et al. (2015), it was demonstrated that diabetes in pregnancy alters the normal 3

development of the hippocampus in the offspring resulted in changes in their behaviours (38, 39). Other researchers have also revealed that hyperglycemia, during the hippocampus development, decreases the maintenance of synaptic plasticity and memory consolidation (16, 28). Although the exact molecular and cellular mechanism by which diabetes during pregnancy affects the development of the brain is still unknown (16, 29, 40), it has clearly shown that maternal hyperglycemia may adversely affect the development of neuronal cells and fetal brain

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structures (12, 41).

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In the previous our studies, we have shown that diabetes during pregnancy led to the various deleterious impacts on the developing hippocampus of the offspring. As in those series of

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studies, in the most cases, there were no significant differences between insulin-treated diabetics

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and the control animals; we concluded that optimal maternal glycaemia control by insulin administration normalized the negative effects of maternal diabetes (25, 28, 36, 42, 43).

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In the present study, we hypothesize that the learning and memory impairment observed in the offspring of diabetic mothers might be due to a decline in neuronal density and volume of the

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hippocampus. In this study, we stereologically estimate the volume and the numerical density of the hippocampal neurons in the two-week-old rats born to diabetic dams.

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Materials and methods

Animals and housing conditions: Thirty virgin female Wistar rats (200–250 g body weight, 6–8 weeks old) were obtained from the Laboratory Animal House of Birjand University of Medical Sciences (Birjand, Iran). Animals were housed in individual cages under temperature controlled standard conditions, 12-hours 4

light/dark cycle with ad libitum access to food pellets, and drinking water. All procedures were conducted by the Animal Welfare Act and approved by the Institutional Laboratory Animal Care and Use Committee of the Birjand University of Medical Sciences, Birjand, Iran. Experimental design All animals were randomly divided into three groups (n=10 in each group):1) Diabetic (STZ-D), 2) Diabetic treated with insulin (STZ-INS), and 3) Control (CON) groups.

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For induction of diabetes, females were injected intraperitoneally (IP) with Streptozotocin (STZ;

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Sigma-Aldrich, 45 mg/kg body weight) freshly dissolved in normal saline. Treatment of diabetic animals was conducted after the verification of diabetes. The diabetic rats in STZ-INS group

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were treated with Protamine-Zinc insulin (NPH) (EXIR Pharmaceutical Company, Iran) twice

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per day (6 U/day, subcutaneously, 2 U at 8:00 AM and 4 U at 5:00 PM) (44-46). The other groups (STZ-D and CON) received both subcutaneous saline injections at the same volume per

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injection of insulin (42). Animals were mated with non-diabetic males overnight starting two days after treatments (35). The presence of a vaginal plug at the following morning was

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designated as day 1 of pregnancy (GD1). Blood glucose concentration (BGC) in animals was measured using a commercially available digital glucometer (BIONIME, Switzerland). Only female animals that are exhibiting BGC > 350 mg/dl on the day of plug observation were used in the present study. At the end of pregnancy, animals were allowed to deliver naturally; the day of

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birth was defined as postnatal day 0 (P0). Newborn rats born to diabetic and Insulin-treated diabetic mothers were fostered onto control mothers to exclude other effects by the milk of diabetic rats and thus enabled to focus only on the environment of the fetal period. Histological studies:

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Two weeks after birth, male offspring were deeply anaesthetized intraperitoneally with chloral hydrate (400mg/kg body weight) and transcardially perfused with 0.9% saline followed by a fixative solution containing 4 % paraformaldehyde and 0.1 M glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and their brains were rapidly dissected, weighted, and then post- immersed in the same fixative for 48 h at 4 °C. The brain tissues were processed by routine histological methods and embedded in paraffin blocks. Serial coronal sections of the brains with 30-μm thickness were

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obtained using a rotatory microtome. For stereological analysis, every 6th section (at the interval

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of 180 µm) including the hippocampus was chosen and stained with Cresyl Violet. Stereological analysis

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The rat brain atlas by Paxinos and Watson was used for the determination of the location of the

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various hippocampal subfields (Fig 1) (47). The volume of the total hippocampus and its subregions were unbiasedly estimated by means of point counting method, using the Cavalier’s

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principle (48, 49). Briefly, a grid of point was superimposed on the images. The points hitting on the total hippocampus and its subfields (CA1, CA2, CA3, DG, and Subiculum) were separately

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counted.

The total volume of the hippocampus and its

subfields

was determined by applying the

following formula:

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Volume = ΣP× d×t×a/p

Where “ΣP” represents the number of points hitting the hippocampus; “d” is equal to the average distance from one section to the next (d= 0.18mm); “t” is the mean section thickness (t=0.03 mm), and “a/p” is equal to the area associated with one point in the grid (50-53). For estimation

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of the volume of the pup’s hippocampal subregions (CA1, CA2, CA3, DG, and Sub), the following formula was used. Vv =∑Q / ∑P×TV In the mentioned formula “∑Q” represents the number of points hitting the hippocampal subfields, ∑P implies the total number of points hitting the total hippocampus and TV was the

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estimated total volume of the hippocampus in our experiment (Fig 2) (54, 55).

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The numerical density and the number of cells within the various hippocampal subfields were separately estimated by an investigator blinded to the protocol treatment, using the optical

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dissector technique(56). This technique eliminates bias in counting as a result of cell size and shape. In brief, cells were counted as they came into focus while scanning through the section. In

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the present study, 8–10 sections, including the hippocampus from each rat per group, were

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subjected to analysis. For each section, 8–10 unbiased counting frames from the various hippocampal sub-regions were sampled in a systematically random fashion (57).

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The preparations were examined under an Eclipse microscope (E200, Nikon, Tokyo, Japan), with a high-numerical-aperture (NA = 1.25) × 60 oil-immersion objectives. Images were transferred to a computer and an electronic microcator with digital readout (MT12, Heidnehain, Traunreut, Germany) using a high-resolution camera (BX51, Japan). The thickness of the

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sections was also measured by focusing on the top of the section, setting the Z-axis to 0, and then refocusing to the bottom of the section and recording the actual thickness (58, 59). The number of cells was counted using computer-generated counting frames (50 µm×50µm for P14). The mean neuronal density of cells in each hippocampal sub-region was estimated as follows: NV=∑Q / [∑P×a(f)×h] 7

Where “∑Q-” is the total number of counted cells within the sampling volume, “∑P” is the number of dissectors, “a/f” is the area of the hippocampus (a/f = 0.001369 mm2) and “h” is the height of the dissector (h = 0.015 mm) (Fig 3). Statistical analysis All results are presented as mean ± SEM. SPSS for Windows was used to perform the statistical

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analysis Results were analyzed using one-way ANOVA, followed by Tukey’s post-hoc test. The

Results

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Maternal and neonatal blood glucose concentrations

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level of statistical significance was set at P < 0.05.

Blood glucose concentrations (BGCs) before, at the end of pregnancy, and in rat neonates (P14)

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of different groups were measured. As shown in table1, the maternal BGC in STZ-D group was

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significantly higher than that in STZ- INS and CON animals (P<0.05). These data confirm that STZ-D rats have remained hyperglycemic throughout gestation. There was no statistical

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difference between the maternal BGC of the STZ-INS and CON groups (P>0.05). Additionally, there was no significant difference between neonatal BGCs in offspring born to the mother of the STZ-D group with STZ-INS and controls (P<0.05, Table 1). Moreover, the body and brain weights of mothers and neonates have also shown in (Table 2). At the end of

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pregnancy, there was a significant difference in the mean body weight of STZ-D group mothers compared to the other groups.

Effects of maternal diabetes on the total volume of the hippocampus

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The total volumes of the left and right hippocampi of the STZ-D group were significantly decreased when compared to other groups (Fig 4. P<0.05).There was no significant difference between the total volume of the right and left sides of the hippocampus in all experimental groups (P>0.05). Effects of maternal diabetes on the volume of hippocampal areas In the hippocampal subfields, the CA1, DG, and subiculum volumes were significantly

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decreased in the STZ-D group compared with the diabetic treated with insulin group and controls

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(P<0.05). However, there were no significant differences in the volume of the CA2, CA3, and subiculum subfields between STZ-D group compared with the control group, and diabetic with

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STZ-INS. (Fig 5, P<0.05).

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Effects of maternal diabetes on the number of cells in the hippocampus

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The numerical density of hippocampal cells was also separately estimated in different sub areas of the hippocampus, and the results are presented in Fig. 3. In all hippocampus sub-regions, maternal diabetes resulted in a significant decrease in the

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number of cells in comparison with two other groups (P≤0.01 each). Our statistics showed no differences in the number of cells between STZ-INS and control group animals (P>0.05 each;

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Fig. 5.).

Discussion:

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In the present study, we evaluated the numerical density of neural cells and the volume of the hippocampus and its subfields in two-week-old rat pups born to diabetic mothers. The Cavalieri’s principle was applied to estimate the hippocampus volumes in the neonatal rats (60, 61). The optical dissector technique is also utilized to determine the neuronal density of cells in the hippocampus, which is a reliable method of cell counting (62).

Since it has been

structure and function, this study only utilized the male offspring.

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demonstrated that sex differentially affects the development and maturation of the hippocampus

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Regarding the fact that, STZ, as a well-known diabetogenic drug, is cleared from the mother’s circulation within 6h, and there were no significant differences in BGCs between control and

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STZ-INS group; it can be concluded that the administration of insulin could control

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hyperglycemic condition, as well, all changes reported in cellular density and hippocampal volume are unlikely to be the direct effects of STZ.

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Unlike insulin, glucose can easily transfer from the maternal blood stream into fetal circulation through the placental barrier. Therefore, maternal hyperglycemia parallels fetal hyperglycemia

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(63-65), which increases the insulin secretion in the fetus (66-69). It was demonstrated that maternal diabetes in pregnancy has detrimental effects on both structural and functional aspects of the offspring’s hippocampus (70). In agreement with previous studies, we found a significant bilateral hippocampal atrophy in diabetic group animals.

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In the present study, we found a bilateral reduction in the numerical density of cells in various hippocampal areas, particularly in CA1 and DG. These results are in consist of earlier studies that showed a lower neuronal density in the cerebrum (71), hippocampus (43, 72), and granular layer of the dentate gyrus in diabetic rats. (73). Moreover, Xue Het et al. (2014) reported a neuronal loss in the hippocampal CA1 region of diabetic animals, 70 days after STZ 10

administration (74). There is evidence demonstrating the extensive range of damage to different regions of the hippocampus in the offspring of diabetic mothers. For example, Lotfi et al. (2016), using TUNEL assay reported a significant increase in apoptotic cells in the hippocampal CA3 field in neonates born to diabetic animals. Moreover, they described that the number of apoptotic cells was insignificantly increased in all hippocampal sub-regions of diabetic group pups when compared to control and STZ-INS group pups at P0, P7, and P14. There was also a slight

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difference either in the total number or in the number of apoptotic cells in the different

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hippocampal sub-fields between the STZ-INS group and controls (30). Besides, in our previous investigation, we showed a significantly higher level of hippocampal DCX (as a marker for

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immature neurons) expression and an increase in the mean number of DCX positive cells in the

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DG of diabetic group male offspring. Nevertheless, the results of immuno- fluorescence staining for NeuN (as a marker for mature neurons) also indicated that the mean number of NeuN

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reactive cells was significantly lower in DG of diabetic group offspring. Besides, there was a significant down-regulation in the hippocampal mRNA expression of NeuN in diabetic pups

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compare to control. Overall, we concluded that diabetes during pregnancy could increase the immature neurons in the hippocampus to ameliorate the adverse effect of maternal diabetes on the maturation of hippocampal neurons.

Taken together, diabetes decreases neurogenesis in the hippocampus. Furthermore, we reported

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that the control of glycemia by insulin is sufficient to prevent the alterations in the expression of neurogenesis markers (42). Although in this study the mechanisms underlying the reductions in the volume and the numerical density of hippocampal cells were not investigated, Nevertheless; it seems that the increases in the neuronal apoptosis and declines in the generation of new neurons in the hippocampus due to maternal diabetes can be considered as a reason for the 11

decrease in volume and neuronal density of the hippocampus of newborns born to diabetic mothers observed in the present study. Our results revealed that there were no significant differences in the neuronal density and volume of hippocampal subfields between the STZ-INS group and controls. Besides, many investigations have proven that insulin has a neuromodulatory effect that controls neurotransmitters release which has an important effect on learning and memory (22), and plays and the function of

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an important role in maintaining neuronal survival, development,

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hippocampus(11, 36). The efficiency of the passive-avoidance memory task can be improved by insulin administration, and on the other hand, insulin receptors in the hippocampus are altered by

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spatial memory training(75).

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Accordingly, this study showed that uncontrolled maternal diabetes induces negative effects hippocampal volume and also decreased in numerical densities of cels in the hippocampuse in

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the first postnatal 2 weeks. These alterations may result in a delay in normal hippocampal development and could be a reason for the structural, motor, behavioral, and cognitive

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abnormalities observed in offspring of diabetic mothers. Further studies are required for exploring the exact mechanism of CNS complications of diabetes in pregnancy and to determine the effects of diabetes during pregnancy on the expression of some related neurotrophic factors in other brain regions, in addition to the importance of the current study’s results.

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In conclusion, this study demonstrated that changes in neural density and hippocampal volume could be considered, at least partly, as a reason for defects in hippocampal development and cognitive impairments observed in offspring born to diabetic rats. Ethical issue:

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All the experiments were conducted according to principal international guidelines, which were approved bythe local ethic committee for Animal Experiments at Birjand University of Medical Sciences, Birjand, Iran (IR.BUMS.REC.1397.051).

The ethic number of this project is IR.BUMS.REC.1397.051

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Figure legends:

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Fig 1: Anatomical boundaries of the hippocampal formation and its subdivisions were distinguished under light microscope according to The Rat Brain Atlas and cell morphology. The hippocampus is composed of two regions: the dentate gyrus (DG) and CornuAmmonis (CA). The CA region is divided into subfields, CA1 contains small pyramidal cells. Field CA2 is characterized by a narrow, dense band of large pyramidal cells and field CA3 by a broad, loose band of large pyramidal neurons

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Fig 2: The estimation of the total volume of the hippocampus and its subfields (CA 1, CA2, CA3, DG and subiculum) using by the Cavalieri’s principle.200µm

Fig 3: Application of optical dissector method for estimation of numerical density and number of neurons in all subfields of the hippocampus (CA 1, CA2, CA3, DG, and subiculum). ×60 19

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Fig 4: Total hippocampal volume (Right and Left) * P < 0.05 compared to controls. # P < 0.05 compared to STZ -INS.

Fig 5: Total volume of hippocampal subregions. The data indicated that maternal diabetes reduced the total hippocampal volume. Moreover, the volume of the CA1, DG, and subiculum in STZ-D group were decreased relative to other groups. * P < 0.05 compared to controls. # P < 0.05 compared to STZ -INS.

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Fig 6: The numerical densities of cells per volume unit in different subfields of the hippocampus. The results showed that maternal diabetes significantly reduced the total number of neurons in the CA1, DG, and subiculum in STZ-D group when compared to other groups. All data must be multiplied in ×103 * P < 0.05 compared to CON. # P < 0.05 compared to STZ -INS.

Table. 1: The level of blood glucose (µg/dl) of the mother and newborn rats. The maternal BGC in STZ-Dgroup

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was significantly higher than that of diabetic treated with STZ- INS as well as CONanimals (P<0.05). These data show that STZ- D rats remained hyperglycemic throughout gestation.

Blood glucose concentration (µg/l)

Groups CON

STZ-D

STZ-INS

100±7.05

501±45.3*#

120±8.8

At the end of pregnancy (GD21)

105±9.9

499±56.4*#

106±6.1

Neonates

111±11.8

102±10.7

99±9.7

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Before pregnancy (GD0)

Values are Mean ± SEM; n = 10. * P < 0.05 compared to controls.

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# P < 0.05 compared to STZ -INS. Table 2: Effect of maternal diabetes and insulin treatment on the body weight and brain weight of neonatal rats. Body and brain weights showed a significant difference between body weight of STZ-D group’s mothers with other groups at the end of pregnancy.

Groups Body weight (G)

STZ-D

STZ-INS

Before pregnancy

281±43.04

255±32.4

260±35.08

At the end of pregnancy

415±22.5

435±31.2*#

388±25.01

Neonate at P14

31.67±1.31

28.45±2.54

41.2±1.34

Brain weight at P14

1.321±0.111

1.520±0.020

1.444±0.033

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CON

* P < 0.05 compared to controls.

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# P < 0.05 compared to STZ -INS.

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