Evaluation of inflammatory mediators in fetal blood and fluids in response to induced endotoxemia in pregnant mares

Author(s)

Marília Alves Ferreira

• Document type: Doctoral Thesis
• Press: Pirassununga.
• Institution: Universidade de São Paulo (USP). Faculdade de Zootecnica e Engenharia de Alimentos (FZE/BT)
• Defense date: 2023-10-11

Examining board members

Renata Gebara Sampaio Dória; Fabiana Fernandes Bressan; Rita de Cassia Campebell; Rafael Resende Faleiros; Claudia Barbosa Fernandes

• Advisor: Renata Gebara Sampaio Dória

Abstract

Systemic inflammatory response syndrome is a consequence of equine acute abdomen. From the activation of the inflammatory cascade, there is the recognition of pathogens and molecular patterns associated with pathogens, through pattern recognition receptors, which detect a specific ligand, such as lipopolysaccharide (LPS). Studies have associated abortions with signs of endotoxemia in mares in the final third of pregnancy, and few studies have been developed for this purpose, especially with sick pregnant mares. This study aims to determine and quantify the presence of inflammatory and anti-inflammatory cytokines, activity of the TLR-4 pattern recognition receptor and the nuclear factor NFκ-B and the metabolomics of blood and fetal fluids, in pregnant mares, during the transitory inflammatory systemic response, induced by intravenous (IV) administration of LPS. Sixteen healthy mares, with 260 to 285 days of pregnancy, mixed breed, aged between 5 and 12 years and body weight between 300 and 450 kg were used. Previous catheterization of the allantois or amniotic cavity was performed, followed by IV infusion of LPS (LPS Group, LPSG; n=8) or 0.9% saline solution (Control Group, CG; n=8) and blood samples were collected and fetal fluids. Hematological and serum biochemistry evaluations, concentrations of the cytokines TNF-α, Interleukin 1 beta (IL1β), Interleukin 6 (IL-6) and interleukin 10 (IL-10) were performed by means of immunoenzymatic assay (ELISA), evaluation of the expression gene expression of TLR-4 receptors and nuclear factor NFκ-B, through real-time PCR, and metabolomics analysis, through liquid chromatography coupled to mass spectrometry. Samples were collected before the beginning of the experiment (T0), after 15 minutes of endotoxemia induction (T15\'), and every one hour, during the first eight hours (T1h, T2h, T3h, T4h, T5h, T6h, T7h, T8h). The pregnancies and placentas were monitored by ultrasonography, with macro and microscopic evaluation of the placenta collected after delivery and neonatal evaluation of the foal being performed. It was verified, in the LPSG, leukopenia in T15\', T1h, T2h, T3h, T4h and T5h in relation to T0h and, smaller means of leukocytes in the LPSG, in T1h, T2h, T3h and T4h, when compared to the CG. There was an increase in the means of urea in the LPSG, in T3h, T4h, T5h, T6h, T7h and T8h, in relation to T0h and an increase in the means of serum creatinine, from T2h to T8h, when compared to T0h. There was an increase in the means of alkaline phosphatase, from T2h to T8h, in the LPSG, compared to T0h, and an increase in the means of total proteins in T6h, when compared to T0h. LPSG IL-6 serum averages increased over time, from T3h to T8h, when compared to T0h, and to the CG. There was an increase in IL-10 means, in the LPSG, from T2h to T7h, in relation to T0h, and to the CG. The means of TNF-α in the LPSG showed an increase in times T1h and T2h, in relation to T0h, and to the CG. There was an increase in gene expression of the TLR4 receptor, in the LPSG, at T3h and T6h, in relation to T0h, differing from the CG at T6h. There was an increase in the expression of the nuclear transcription factor NFκ-B in the LPSG, in relation to the CG, at T6h. With regard to fetal fluids, the means of IL-1β, in LPSG, showed an increase in T6h and T12h, compared to T0h. The LPSG showed higher means of IL-1β, at T6h and T12h, when compared to the CG. In LPSG, there was an increase in TNF-α means at T12h, when compared to T0h. It was verified, in T3h, higher means of IL-10 in the LPSG, in relation to the CG. The results of plasma metabolomics and allantoic fluid demonstrate that LPSG has distinct metabolites and different signal intensity from the metabolites found in the CG, with a total of 309 and 626 important metabolites in plasma and allantoic fluid, respectively, which allow the separation between groups. At the end of the study, 57% of abortions and 43% of deliveries were observed in the CG and 87.5% of abortions and 12.5% of premature births in the LPSG, with ultrasonographic and macroscopic changes characteristic of placentitis, with microscopic inflammatory changes. Born foals presented APGAR scores of 7 to 8. Kidney, liver and lung samples from the abortions showed inflammatory and degenerative characteristics. It is concluded that there is influence of maternal endotoxemia on the fetus-placental unit, evidenced by the elevation of inflammatory cytokines in fetal fluids and metabolite profile, which can characterize endotoxemia in pregnant mares. Catheterization of the allantoic and amniotic cavities is a feasible technique with a low degree of difficulty and poses no risk to the mare, although it may cause abortion and is not indicated for use in clinical routines. (AU)

• FAPESP's process: 19/22059-3 - Evaluation of inflammatory mediators in blood and fetal fluids in response to induced endotoxemia in pregnant mares
• Grantee: Marília Alves Ferreira
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)