Adult rat jejunum and liver histological measurements and gene expression after perinatal exposure 5-hydroxytryptophan and tranylcypromine

Description

Maintaining serotonin (5HT) homeostasis is vital for physiological processes in the central nervous system and peripheral tissues. Hyperserotonemia, a measurable sign of 5HT homeostasis disruption that can be caused by 5HT-directed treatments for psychiatric and gastrointestinal disorders, has unresolved implications for long-term 5HT equilibrium in the periphery. This study investigates perinatal 5HT imbalance effects on jejunum and liver in adult rats. Hyperserotonemia was induced by 5-hydroxytryptophan (5HTP) or tranylcypromine (TCP) treatment. At postnatal day 70, jejunum and liver samples were analyzed. Compared to controls, 5HTP and TCP-treated rats showed reduced 5HT-producing cells and decreased jejunum 5HT-synthesizing enzyme expression. Additionally, there was increased 5HT transporter expression with hepatocyte karyomegaly, more pronounced in TCP-treated animals. This study reveals lasting cellular and molecular changes due to perinatal 5HT imbalance, potentially impacting 5HT availability in the periphery. The rat model links developmental serotonin abnormalities to adult 5HT-related changes, offering insights into susceptibility to behavioral and metabolic disorders. It serves as a model for exploring adverse effects of prenatal exposure to 5HT enhancers in humans. The study involved a two-step chronic treatment of animals with either of the two 5HT enhancers—5-hydroxytryptophan (5HTP) and tranylcypromine (TCP)—or saline. In the first step, pregnant females (3 with each 5HT enhancer and 2 with saline) were treated from gestational day 12 until parturition. In the second step, pups (13 with TCP, 13 with 5HTP, and 11 with saline) were treated from postnatal day (PND) 1 until PND21. At PND70, blood, jejunum, and liver samples were collected from all experimental and control rats. Serum 5HT concentrations were measured with ELISA, and jejunum and liver tissues were examined histomorphologically. The relative expression of genes related to the serotonin pathway proteins, including tryptophan hydroxylase 1 (Tph1) and vesicular monoamine transporter 1 (Vmat1) responsible for serotonin synthesis and storage in the jejunum, and serotonin transporter (5HTt) and monoamine oxidase A (MaoA) responsible for 5HT elimination in the liver, was determined via quantitative PCR.

Steps to reproduce

On postnatal day 70 ± 1, all 37 rats were euthanized under isoflurane anesthesia. Blood samples were collected according to Blazevic et al. (2015). For mRNA analysis, approximately 85 mg of liver and 65 mg of jejunum tissue were promptly cut, washed in cold saline, and frozen in liquid nitrogen. Samples for histological processing were fixed in 10% neutral formalin, dehydrated, cleared, and embedded in Paraplast embedding media. Gene expression: Samples were homogenized and frozen until further processing. RNA was isolated with RNAqueous-4PCR kit, eliminating genomic DNA as per instructions. RNA quality was assessed through agarose gel electrophoresis and spectrophotometry. mRNA was reverse-transcribed using MuLV reverse transcriptase and oligo dT primers. Reverse transcription efficacy was confirmed through endpoint PCR. Relative expression of Tph1, MaoA, Vmat, and 5HTt was evaluated via qPCR using TaqMan gene expression master mix. Rat ACTB or GAPDH served as endogenous control reference genes in a duplex setup. The qPCR setup included a two-minute incubation at 50 °C, followed by 10 minutes at 95 °C, and 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Relative gene expression was calculated following the Pfaffl method with efficiency correction. Experiments adhered to MIQE guidelines. Histology: Cross sections (5–7 µm thick) were stained with hematoxylin–eosin (HE) for general histology and Masson–Fontana method for neuroendocrine argentaffin cells in the small intestine. Tissue samples were coded and independently studied in a blinded fashion, with 1–2 randomly selected sections for each rat. The entire digestive tract surface (excluding the lumen) was measured, and positive cells were counted in a single jejunum cross-section, distinguishing between surface and crypt epithelial areas. Results were expressed as the average number of argentaffin cells per 1 mm of reference space, excluding reactions in lymphocytes, erythrocytes, and Paneth cells. For quantifying argentaffin granules, cells were examined at 1000× magnification, assigning values from 1 to 3 for stain intensity, with results averaged for each animal. Additionally, mucosa thickness, mucosal epithelial cell height, and outer muscle wall thickness were measured at 5 randomly selected test fields at 100× magnification. Liver sections were stained with periodic acid–Schiff (PAS). Morphometric analysis of 10 randomly chosen test fields (at 200× magnification) per animal was conducted on 34 liver samples stained with PAS. Measures included the width of 10 hepatocytes, width of 10 hepatocyte nuclei, diameter of 10 sinusoids, and minimum and maximum diameter of a central vein in the pericentral zone around the central vein. All morphometric assessments were performed using a computerized image analysis system with a light microscope, digital camera, and image analysis software. - Blazevic et. al. J. Physiol. Pharmacol. 2015, 66, 529–537.

Institutions

Categories

Funding

Licence