Chronic Intra-Uterine Ureaplasma parvum Infection Induces Injury of the Enteric Nervous System in Ovine Fetuses

Abstract

Background: Chorioamnionitis, inflammation of the fetal membranes during pregnancy, is often caused by intra-amniotic (IA) infection with single or multiple microbes. Chorioamnionitis can be either acute or chronic and is associated with adverse postnatal outcomes of the intestine, including necrotizing enterocolitis (NEC). Neonates with NEC have structural and functional damage to the intestinal mucosa and the enteric nervous system (ENS), with loss of enteric neurons and glial cells. Yet, the impact of acute, chronic, or repetitive antenatal inflammatory stimuli on the development of the intestinal mucosa and ENS has not been studied. The aim of this study was therefore to investigate the effect of acute, chronic, and repetitive microbial exposure on the intestinal mucosa, submucosa and ENS in premature lambs. Materials and Methods: A sheep model of pregnancy was used in which the ileal mucosa, submucosa, and ENS were assessed following IA exposure to lipopolysaccharide (LPS) for 2 or 7 days (acute), Ureaplasma parvum (UP) for 42 days (chronic), or repetitive microbial exposure (42 days UP with 2 or 7 days LPS). Results: IA LPS exposure for 7 days or IA UP exposure for 42 days caused intestinal injury and inflammation in the mucosal and submucosal layers of the gut. Repetitive microbial exposure did not further aggravate injury of the terminal ileum. Chronic IA UP exposure caused significant structural ENS alterations characterized by loss of PGP9.5 Heymans et al. Chorioamnionitis Induces ENS Alterations and S100β immunoreactivity, whereas these changes were not found after re-exposure of chronic UP-exposed fetuses to LPS for 2 or 7 days. Conclusion: The in utero loss of PGP9.5 and S100β immunoreactivity following chronic UP exposure corresponds with intestinal changes in neonates with NEC and may therefore form a novel mechanistic explanation for the association of chorioamnionitis and NEC.

Chorioamnionitis induces enteric nervous system injury: effects of timing and inflammation in the ovine fetus

“…Whether such an accelerated maturation is sufficient to fully compensate for the identified loss of neurons Table 2 Immune cells count in the mucosal layer Control (n = 6) 5 h LPS (n = 6) 12 h LPS (n = 7) 24 h LPS (n = 7) 2d LPS (n = 6) 4d LPS (n = 6) 8d LPS (n = 7) 15d LPS (n = 6) remains to be elucidated. These findings combined with the unaltered PGP9.5-positive and doublecortinpositive surface area after 2 and 7 days of IA LPS exposure in a previous study (Heymans et al 2020), show that the ENS changes found are time-dependent and may recover following prolonged intrauterine inflammation. Interestingly, in a previous study, a similar loss of mature neurons was observed after chronic IA exposure to UP, indicating that different inflammatory triggers can induce similar ENS damage (Heymans et al 2020).…”

“…These findings combined with the unaltered PGP9.5-positive and doublecortinpositive surface area after 2 and 7 days of IA LPS exposure in a previous study (Heymans et al 2020), show that the ENS changes found are time-dependent and may recover following prolonged intrauterine inflammation. Interestingly, in a previous study, a similar loss of mature neurons was observed after chronic IA exposure to UP, indicating that different inflammatory triggers can induce similar ENS damage (Heymans et al 2020). Enteric glial cells are important for neuronal maintenance, survival, and function (De Giorgio et al 2012), and are capable of generating enteric neurons in response to injury (Joseph et al 2011;Laranjeira et al 2011).…”

“…In addition, enteric glia respond, in a manner similar as reactive astrogliosis in the central nervous system, to ENS injury and inflammation by changing both their morphology and their expression of key proteins such as GFAP (Boesmans et al 2015 ; Rosenbaum et al 2016 ). The neuronal loss in the myenteric plexus after 4 days of LPS exposure is accompanied with a reduced S100β-positive surface area, likely representing a loss of glial cells and/or loss of S100β immunoreactivity within glial cells, as was earlier described during chronic IA UP exposure (Heymans et al 2020 ). Interestingly, this loss of neurons and glial cells is preceded by an increased myenteric plexus GFAP immunoreactivity after 2 days of LPS exposure.…”

“…c GFAP-positive surface area in the myenteric plexus was increased in animals exposed to 2 days of IA LPS. * p < 0.05 compared to control in fetal lambs that were chronically IA exposed to UP (Heymans et al 2020). Moreover, inflammation driven pathological changes of the ENS are more often found in the myenteric plexus than in the submucosal plexus (De Giorgio et al 2004).…”

“…Recently, we have shown in a preclinical ovine model that chronic chorioamnionitis is associated with significant structural ENS abnormalities (Heymans et al 2020 ). Importantly, these alterations corresponded with those found in infants with NEC, indicating that ENS changes following chorioamnionitis may predispose to later NEC development (Heymans et al 2020 ). Since inflammation is a dynamic process and the vulnerability of the fetus to injurious exposure during intra-uterine development varies, ENS alterations in response to inflammation can be time-dependent.…”