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Yasunori Masubuchi, Takaharu Tanaka, Rena Okada, Yuko Ito, Junta Nakahara, Satomi Kikuchi, Yousuke Watanabe, Toshinori Yoshida, Robert R. Maronpot, Mihoko Koyanagi, Shim-mo Hayashi, and Makoto Shibutani
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Hypothyroidism during the developmental stage induces disruption of hippocampal neurogenesis in later life, as well as inducing oxidative stress in the brain. The present study investigated the preventive effect of co-exposure to an antioxidant on disruptive neurogenesis induced by developmental exposure to anti-thyroid agent in rats. For this purpose, we used two antioxidants, α-glycosyl isoquercitrin (AGIQ) and α-lipoic acid (ALA). Mated female Sprague Dawley rats were either untreated (control) or treated with 12 ppm 6-propyl-2-thiouracil (PTU), an anti-thyroid agent, in drinking water from gestational day 6 to postnatal day (PND) 21, the latter group being subjected to feeding basal diet alone or diet containing AGIQ at 5,000 ppm or ALA at 2,000 ppm during PTU exposure. On PND 21, PTU-exposed offspring showed reductions in a broad range of granule cell lineage subpopulations and a change in the number of GABAergic interneuron subpopulations. Co-exposure of AGIQ or ALA with PTU altered the transcript levels of many genes across multiple functions, suggestive of enhancement of synaptic plasticity and neurogenesis. Nevertheless, immunohistochemical results did not support these changes. PTU exposure and co-exposure of AGIQ or ALA with PTU did not alter the hippocampal lipid peroxidation level. The obtained results suggest a possibility that thyroid hormone depletion itself primarily disrupts neurogenesis and that oxidative stress may not be involved in the disruption during development. Transcript expression changes of many genes caused by antioxidants may be the result of neuroprotective actions of antioxidants rather than their antioxidant activity. However, no preventive effect on neurogenesis suggested impairment of protein synthesis via an effect on mRNA translation due to hypothyroidism.

Introduction

The hippocampus is an important temporal lobe brain structure involved in cognition, learning, and memory. The hippocampus postnatally generates new neurons within the subgranular zone (SGZ) of the dentate gyrus (DG), which is termed “adult neurogenesis” (Fig. 1)12. This neuronal production consists of multistep processes, including a number of developmental phases, such as self-renewal of neural stem cells, the facilitation of continued division of precursor cells to produce new granule cells, and subsequent differentiation and migration of these new cells into the granule cell layer (GCL)12. In the hilus of the DG, subpopulations of γ-aminobutyric acid-ergic (GABAergic) interneurons innervate granule cell lineage populations to control neurogenesis in the SGZ23. In addition to GABAergic neuronal inputs, various types of neurons outside the SGZ also create a synaptic connection with neurons in the DG, such as glutamatergic neurons in the entorhinal cortex providing axonal projections to the DG4 and cholinergic neurons originating from the septal nucleus and nucleus of the diagonal band of Broca innervating neurons in the hilus of DG4. Glutamatergic inputs to the SGZ are important for maintaining proper proliferation and differentiation of the granule cell lineage subpopulations3.

Fig. 1. Overview of the hippocampal formation of a male rat stained with hematoxylin and eosin. The number of cells in the hilus of the dentate gyrus (as enclosed by the dotted line) displaying immunoreactivity for parvalbumin (PVALB), reelin (RELN), calbindin-D-29K (CALB2), somatostatin (SST), or neuronal nuclei (NeuN) was counted and normalized for the unit area. Only small-sized cells with positive immunoreactivity for these antigens were counted, and larger Cornu Ammonis (CA) 3 neurons were excluded. (Inset) Higher magnification of the granule cell layer (GCL) and subgranular zone (SGZ). The numbers of immunoreactive cells for glial fibrillary acidic protein (GFAP); SRY box 2 (SOX2); T-box brain 2 (TBR2); doublecortin (DCX); NeuN; activity-regulated cytoskeleton-associated protein (ARC); Fos proto-oncogene, AP-1 transcription factor subunit (FOS); and cyclooxygenase 2 (COX2) and the number of proliferating cells or apoptotic cells were measured in the SGZ and/or GCL. Magnification ×40; bar 200 µm. (Inset) Higher magnification of the granule cell layer. Magnification ×400; bar 50 µm.

Thyroid hormones are crucial for brain development during fetal and neonatal periods. They play important roles in neuronal proliferation and migration, neuritogenesis, synaptogenesis, and myelinogenesis5. Previous studies have shown that rat developmental hypothyroidism causes aberrant brain growth involving diverse cellular populations and also impairs inherent brain structures and functions5. Hypothyroidism impairs neuronal migration and results in subcortical band heterotopia in the corpus callosum56, as well as white matter hypoplasia with suppression of both axonal myelination and oligodendrocytic accumulation7. It has been considered that maternal hypothyroidism is associated with autism spectrum disorders (ASD)5. Multiple studies have highlighted the involvement of key processes, such as those including neurogenesis, neurite growth, synaptogenesis, and synaptic plasticity, in the pathophysiology of neurodevelopmental disorders, such as ASD8. Therefore, an experimental induction of developmental hypothyroidism could provide a reasonable model for ASD9.

Oxidative stress is defined as an imbalance between reactive oxygen species (ROS) production and the antioxidant defense system in an organism and is involved in various disorders such as neurodegenerative diseases and malignant tumors. Neural injury in the central and peripheral nervous system caused by some kinds of neurotoxicants is considered to be related to the induction of oxidative stress, but it is unclear how neurotoxicants cause oxidative stress and neurotoxicity. Importantly, SGZ cells in the DG generate ROS, because these cells have a high cellular activity for proliferation and differentiation requiring high oxygen demand10. With regard to the effect of hypothyroidism on brain tissues, induction of oxidative stress has been reported in the rat hippocampus as a result of administration of an anti-thyroid agent during the developmental stage or adult stage1112. Furthermore, co-exposure of extracts of a medicinal plant, Nigella sativa, with an anti-thyroid agent, 6-propyl-2-thiouracil (PTU), from the gestation period to adult stage in rats reduces apoptosis in the hippocampal DG, the Cornu Ammonis region (CA) 1 and CA3 areas, as compared with PTU exposure alone13. Therefore, there is a possibility that developmental exposure to an antioxidant may prevent hypothyroidism-related disruption of hippocampal neurogenesis induced by administration with an anti-thyroid agent during development in rats.

The present study was performed to clarify whether developmental exposure to an antioxidant has a potential to prevent hypothyroidism-related disruption of hippocampal neurogenesis induced by administration with PTU during development in rats. For this purpose, we used two antioxidants, α-glycosyl isoquercitrin (AGIQ) and α-lipoic acid (ALA), in the present study. AGIQ, also known as enzymatically modified isoquercitrin, is a flavonol glycoside derived by enzymatic glycosylation of rutin. AGIQ is a mixture of quercetin glycoside, consisting of isoquercitrin and its α-glucosylated derivatives, with 1–10 or more of additional linear glucose moieties and has greater water solubility and bioavailability14. AGIQ has been reported to exert anti-oxidative15, anti-inflammatory16, anti-hypertensive17, anti-allergic18 and tumor suppressive151920 properties. ALA, a natural compound that is chemically named 5-(1,2-dithiolan-3-yl)pentanoic acid and is also known as thioctic acid21, is another metabolic antioxidant. In addition to direct antioxidant activity, ALA and its endogenous counterpart dihydrolipoic acid (DHLA), which is rapidly formed after uptake into the body’s cells, contributes to the nonenzymatic regeneration of reduced glutathione, vitamin C, vitamin E, and coenzyme Q10 in vivo22. Moreover, DHLA stimulates glutathione synthesis by enhancing cellular cysteine uptake. As DHLA is a supplier of reducing equivalents for the regeneration of detoxification enzymes, it is capable of supporting repair of oxidative damage22. ALA has been reported to prevent or ameliorate several ailments such as cardiovascular diseases, diabetic complications including retinopathy and neuropathy, and hypertension because of its antioxidant properties23. It is known that once absorbed in the body, AGIQ and ALA can pass the blood-brain barrier and be distributed in the brain2425. We have recently found that continuous AGIQ exposure from the developmental stage can facilitate fear extinction learning associated with enhancement of synaptic plasticity at the adult stage in rats26. Experimentally, ALA has been shown to ameliorate brain oxidative injury induced by methionine and choline deficiency27. Therefore, if oxidative stress is involved in aberrant neurogenesis in the hippocampal DG induced by developmental hypothyroidism, there is a possibility that co-exposure to AGIQ or ALA would suppress aberrant neurogenesis in the hippocampal DG.

Materials and Methods

Chemicals and animals

PTU (purity >99%; CAS No. 51-52-5) was purchased from MilliporeSigma (St. Louis, MO, USA). AGIQ (purity >97%) was supplied by San-Ei Gen F.F.I. Inc. (Osaka, Japan). DL-ALA (purity ≥99%; CAS No. 1077-28-7) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Fifty mated female Slc:SD rats were purchased from Japan SLC, Inc. (Hamamatsu, Japan) at gestational day (GD) 1, where GD 0 was the day of appearance of a vaginal plug. Mated female rats were individually housed in polycarbonate cages with paper bedding until postnatal day (PND) 21, where PND 0 was defined as the day of delivery. Animals were maintained in an air-conditioned animal room (temperature, 23 ± 2°C; relative humidity, 55 ± 15%) with a 12-h light/dark cycle. Mated female rats were allowed to access to powdered basal diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water ad libitum until the start of developmental exposure to PTU with or without exposure to AGIQ or ALA. Offspring were weaned on PND 21 and thereafter reared three to five animals per cage and provided with powdered basal diet (CRF-1) and tap water ad libitum.

Experimental design

Mated female rats were randomly divided into four groups by stratified randomization according to the body weight on GD 5; 14 rats were fed basal diet and tap water (untreated controls), 12 rats were fed basal diet and water containing 12 ppm PTU (PTU alone), 12 rats were fed diet containing 5,000 ppm AGIQ and water containing 12 ppm PTU (PTU + AGIQ) or 12 rats were fed diet containing 2,000 ppm ALA and water containing 12 ppm PTU (PTU + ALA) (Fig. 2). Animals were treated from GD 6 to day 21 post-delivery with PTU with or without AGIQ or ALA. Based on a previous study that showed apparent aberrations in neuronal development in the hippocampal structure in offspring28, the PTU dose was set at 12 ppm. The chosen dosages of AGIQ and ALA have both been shown to suppress the promotion of hepatic preneoplastic lesions in rats1920.