Postnatal Development of the Amygdala of Rat Offspring after Prenatal Exposure to Valproic Acid: A Histological and Morphometric Study
Authors: Nagiub Nahla Mohammed , Elrakhawy Fatma Ibrahim , El-Nefiawy Nagwa Ebrahim , Yehia Moustafa Ahmed , Ebrahim Sara Shawky 
Published: August 2023

Abstract

Background
The amygdala has a major role in emotion regulation, social behavior development and reward learning. Being subject to several factors makes it vulnerable to numerous neurodevelopmental disorders.

Aim of the Work
The aim of the current work was to study the structural and morphometric changes of the amygdala of rat's offspring following maternal exposure to valproic acid during pregnancy and to compare it with the control.

Material and methods
20 adult female and 10 adult male Sprague dawley albino rats weighing 150-200 g body weight were used in the current study. After induction of pregnancy, pregnant rats were divided into two groups 6 in each group; control group (I) and valproic acid treated group (II). Pregnant rats received one ml of saline in group (I) or 500 mg/kg BW valproic acid dissolved in one ml saline in group (II) by oral gavage on day 11-13 of pregnancy. Pregnant rats were observed till end of pregnancy and four male newborn rats were sacrificed at each time point of the experiment from the two groups (week1, 2, &3). Brains were extracted and processed for paraffin blocks and sectioned then stained with H&E, cresyl violet stains and immunohistochemically stained for GFAP antibody. Image analysis &morphometry were performed on stained sections for; number of neurons, number of oligodendrocytes, surface area of basal nucleus of amygdala and density of GFAP staining.

Results
On examination of H&E-stained sections of control groups during the first three weeks postnatally, the amygdala contained mainly three types of cells: pyramidal cells, oligodendrocytes, and astrocytes. Pyramidal cells had basophilic cytoplasm with large rounded open face nuclei and prominent nucleoli. Some pyramidal cells showed apical dendrites. The oligodendrocytes were seen to have rounded nuclei with condensed chromatin surrounded by thin cytoplasmic ring. Astrocytes possessed nuclei with pale vesicular chromatin pattern less dense than that of oligodendrocytes and showed relatively small indistinct nucleoli. Morphometric studies using image analysis had recorded statistically non-significant changes in the number of neurons during development, however concerning oligodendrocytes there was a statistically significant increase in their number in week 2 and 3 when compared to week 1. Using Nissl staining (cresyl violet) it demonstrated negative or very weak staining in the first week postnatally, the staining becomes more obvious in the next 2 weeks postnatally. GFAP immune stained sections of the amygdala of the 3 weeks revealed mild positive immune reaction. There was no statistically significant difference regarding density of staining across the three weeks. The surface area of the basal nucleus of amygdala of the control groups showed statistically significant increase during development across the three weeks.
On examination of H&E-stained sections of VPA treated groups during the first three weeks postnatally, numerous cells with pyknotic deeply basophilic stained nuclei were seen. Morphometric studies on the same groups using image analysis recorded statistically significant decrease in neurons and oligodendrocytes number when compared to control. On examination of the cresyl violet stained sections, there were numerous darkly stained degenerating cells that did not reveal the presence of Nissl granules denoting chromatolysis. On examining GFAP immune stained sections of the amygdala, week 2 and 3 postnatally showed strong positive immune reaction. The surface area of the basal nucleus of amygdala of VPA treated groups of weeks 1 showed a highly significant decrease when compared to week 1 of control groups, however the surface area of the amygdala of week 2 and 3 showed an increase more than the increase recorded in the control groups. The increase in the surface area was non-significant in week 2 but highly significant in week 3.

Conclusion
Results of the present experimental study demonstrated the developmental structural changes of the rat amygdala across the first three weeks of postnatal life. The study also explored the devastating effect of the prenatal exposure to VPA on the structure of the amygdala leading to neuronal death and gliosis particularly in the third week of age postnatally.