Author: Greta J E Gnanamanickam

Gnanamanickam, Greta J E, 2015 CHANGES TO THE INNERVATION OF THE RAT UTERUS DURING PREGNANCY AND POST-PARTUM, Flinders University, School of Medicine

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Pre-eclampsia is a hypertensive disorder affecting 5 to 8% of all pregnancies. One of the main characteristics of pre-eclampsia is a reduction in blood flow to the placenta and other maternal organs. Because sympathetic and parasympathetic nerves control blood vessel diameters, I examined the innervation of the uterus during pregnancy and post-partum using whole mount preparations. Whole mounts provide a complete picture of the innervation of all layers of the uterus compared to sections where only small samples can be studied. In my Master’s thesis, I showed that uterine horns from rats at 20 day (E20) of a 21-22 day pregnancy have almost no sympathetic, parasympathetic or sensory nerves. In non-pregnant uterine horns, all uterine layers and uterine blood vessels receive significant sympathetic, parasympathetic and sensory innervation.

In this project, I examined and quantified the loss of uterine sympathetic, parasympathetic and sensory nerves during late pregnancy and their reappearance during the first 4 weeks after delivery. Immunoreactivity for tyrosine hydroxylase (TH) and neuropeptide Y (NPY) were used as markers for sympathetic nerves; vesicular acetylcholine transporter (VAChT), for parasympathetic nerves; and calcitonin gene-related peptide (CGRP), for sensory nerves. I assessed uterine horns from non-pregnant rats at oestrus, rats at pregnancy days E14, E16, E18 and E20 and rats at post-partum (P) days P1, P3, P5, P7, P10, P14 and P28.

In pregnant uterine horns, there was a steady decline in the density of TH-, NPY-, VAChT- and CGRP-immunoreactive axons as pregnancy progressed until innervation had almost completely disappeared near term. By E18, blood vessels were scarcely innervated. By E20 there were only rare axons around blood vessels and virtually none deep in the endometrium or in the longitudinal muscle. Quantification using an unbiased stereological method showed that the density of TH-, CGRP- and VAChT-immunoreactive axons had decreased by over a 99.9% in the E20 uterus compared to the uterus at oestrus. Re-innervation began immediately after delivery; rare TH-, NPY-, VAChT- and CGRP-immunoreactive axons were present in uterine horns at P1. There was a steady increase in the number of TH, NPY, VAChT and CGRP-immunoreactive axons between P1 and P28. Axons with growth cones were abundant in P3 and P5 uterine horns; rare uterine axons still had growth cones at P28. The densities of uterine nerves at P28 were still lower than in the uterus at oestrus, indicating that even at 4 weeks post-partum uterine innervation had not returned to normal.

To investigate a possible mechanism underlying uterine innervation changes, I compared expression of microRNAs extracted from rat major pelvic ganglia (MPG), the source of uterine sympathetic and parasympathetic innervation from rats at oestrus, E20 pregnant rats and P5 and P14 post-partum rats. MicroRNA samples were analysed by microarrays and real time relative quantitation reverse transcription PCR. Expression levels of microRNAs in the intact MPG did not differ among any of the time points examined.

This thesis describes the time course of uterine sympathetic, parasympathetic and sensory denervation during pregnancy and re-innervation post-partum using whole mount preparations and presents the first reliable quantitative assessment of these changes. The molecular biological studies suggest that microRNAs from the MPG may not be involved in uterine innervation changes. The immunohistochemical data will provide a foundation for future studies on the involvement of uterine nerves in pre-eclampsia.

Keywords: uterine innervation, pregnancy, post-partum

Subject: Neuroscience thesis

Thesis type: Doctor of Philosophy
Completed: 2015
School: School of Medicine
Supervisor: Prof. Ida Llewellyn-Smith