Uterine Inflammation Changes the Expression of Cholinergic Neurotransmitters and Decreases the Population of AChE-Positive, Uterus-Innervating Neurons in the Paracervical Ganglion of Sexually Mature Gilts

Abstract

The focus of this study was based on examining the impact of endometritis on the chemical coding of the paracervical ganglion (PCG) perikaryal populations supplying pig uterus. Four weeks after the injection of Fast Blue retrograde tracer into uterine horns, either the Escherichia coli (E. coli) suspension or saline solution was applied to both horns. Laparotomy treatment was performed for the control group. Uterine cervices containing PCG were extracted on the eighth day after previous treatments. Subsequent macroscopic and histopathologic examinations acknowledged the severe form of acute endometritis in the E. coli-treated gilts, whereas double-labeling immunofluorescence procedures allowed changes to be analyzed in the PCG perikaryal populations coded with vesicular acetylcholine transporter (VAChT) and/or somatostatin (SOM), vasoactive intestinal polypeptide (VIP), a neuronal isoform of nitric oxide synthase (nNOS), galanin (GAL). The acetylcholinesterase (AChE) detection method was used to check for the presence and changes in the expression of this enzyme and further confirm the presence of cholinergic perikarya in PCG. Treatment with E. coli resulted in an increase in VAChT+/VIP+, VAChT+/VIP-, VAChT+/SOM+, VAChT+/SOM-, VAChT+/GAL- and VAChT+/nNOS- PCG uterine perikarya. An additional increase was noted in the non-cholinergic VIP-, SOM- and nNOS-immunopositive populations, as well as a decrease in the number of cholinergic nNOS-positive perikarya. Moreover, the population of cholinergic GAL-expressing perikarya that appeared in the E. coli-injected gilts and E. coli injections lowered the number of AChE-positive perikarya. The neurochemical characteristics of the cholinergic uterine perikarya of the PCG were altered and influenced by the pathological state (inflammation of the uterus). These results may indicate the additional influence of such a state on the functioning of this organ.

Keywords: chemical coding; cholinergic innervation; endometritis; immunocytochemistry; paracervical ganglion; pig; uterine neurons.

Figure 1

The populations (expressed as percentages, mean ± SEM) of uterine perikarya expressing vesicular acetylcholine transporter (VAChT) and/or vasoactive intestinal polypeptide (VIP) (A), VAChT and/or somatostatin (SOM) (B), VAChT and/or galanin (GAL) (C), and VAChT and/or neuronal isoform of nitric oxide synthase (nNOS) (D), as well as those without these substances in the PCG of gilts from the control (white bars), saline (grey bars), and E. coli (black bars) groups. Data are expressed as percentages of the total population of uterine perikarya stained for two substances in each group, accepted as 100%. * p < 0.05, ** p < 0.01, and *** p < 0.001 show differences between all groups for the same population of uterine perikarya.

Figure 2

Micrographs demonstrating the presence of VAChT (B,F,J,N), VIP (C,G), and SOM (K,O) in the PCG uterine perikarya of gilts from the saline (A–D), control (I–L), and E. coli (E–H,M–P) groups. The arrowhead indicates a Fast Blue (FB)-positive neuron, a perikaryon immunoreactive to VAChT, VIP, and a perikaryon immunoreactive to SOM. The double arrow indicates an FB-positive uterine neuron expressing VAChT and VIP and VAChT and SOM. The arrow indicates an FB-positive perikaryon expressing SOM. The photographs (D,H,L,P) were made using the digital superimposition of three color channels: FB-positive (blue), VAChT-positive (red), and SOM- or VIP-positive (green). One VAChT and VIP immunoreactive uterine neuron is visible in the gilt of the saline group (A–D). In the PCG of the E. coli group, an elevated number of perikarya expressing these substances are visible (E–H). One perikaryon expressing SOM and VAChT is present in the ganglion of the control group (I–L). In the E. coli group, two perikarya expressing both of these substances are observed in the PCG (M–P).

Figure 3

Micrographs demonstrating the presence of VAChT (B,F,J,N), GAL (C,G), and nNOS (K,O) in the PCG uterine perikarya of gilts from the control (A–D), saline (I–L), and E. coli (E–H,M–P) groups. The arrowhead indicates a Fast Blue (FB)-positive neuron, a perikaryon immunoreactive to VAChT, a neuron immunoreactive to GAL, as well as an nNOS-immunoreactive perikaryon. The double arrow indicates an FB-positive uterine neuron expressing VAChT and GAL/nNOS, whereas the arrow shows an FB-positive and GAL/nNOS immunoreactive neuron. The photographs were made using the digital superimposition of three color channels: FB-positive (blue), VAChT-positive (red), and nNOS- or GAL-positive (green). One perikaryon expressing GAL but not VAChT is present in the ganglion of the control group (A–D). In the E. coli group, a perikaryon expressing these substances is observed in the PCG (E–H). Two VAChT and nNOS immunoreactive uterine neurons are visible in the gilt of the saline group (I–L). In the PCG of the E. coli group, a decreased number of perikarya expressing these substances are visible (M–P).

Figure 4

The numbers of both FB- and VAChT-positive neurons positively stained for AChE (mean ± SEM) in the PCG counted in the gilts from control (CON), saline (SAL), and E. coli (E. coli) groups (* p < 0.05 and ** p < 0.01 show the differences between groups).

Figure 5

Micrographs demonstrating the presence of uterine, VAChT-positive (A,C), and AChE (B,D) expressing perikarya in the PCG of gilts from the saline (A,B) and E. coli (C,D) groups. The double arrow indicates an FB-positive neuron expressing VAChT. The arrow indicates an AChE-positive, whereas the arrowhead indicates an AChE-negative perikaryon. Two uterine neurons expressing VAChT and AChE are visible in the gilt of the saline group (A,B). A decreased number of perikarya expressing AChE are present in the PCG of the bacteria-treated group (C,D).