Ropivacaine Activates Multiple Proapoptotic and Inflammatory Signaling Pathways That Might Subsume to Trigger Epidural-Related Maternal Fever
Authors: Wohlrab Peter , Boehme Stefan , Kaun Christoph , Wojta Johann , Spittler Andreas , Saleh Leila , Knöfler Martin , Markstaller Klaus , Klein Klaus U. , Tretter Verena 
Published: September 2019

BACKGROUND:
Epidural-related maternal fever (ERMF) is an adverse effect of epidural analgesia during labor and is associated with perinatal and neonatal morbidity. Local anesthetics have been proposed to trigger ERMF via sterile inflammation. Ropivacaine is currently the most frequently used epidural anesthetic and considered least toxic. This study investigates molecular effects of ropivacaine on human umbilical vein endothelial cells (HUVECs) as model system for endothelial cells and human placental trophoblasts (TBs), compares the effects to the putative anti-inflammatory lidocaine and investigates the partially alleviating impact of the anti-inflammatory corticosteroid dexamethasone.

METHODS:
HUVECs and TBs were exposed to ropivacaine (35 μM–7 mM) or lidocaine (21 mM) with or without dexamethasone (1 μM). AnnexinV/propidium iodide staining and lactate dehydrogenase release were used to analyze apoptosis and cytotoxicity. Proinflammatory interleukins-6 (IL-6) and IL-8 as well as prostaglandin E2 (PGE2) were measured by enzyme-linked immunosorbent assay (ELISA), while activation of signaling pathways was detected by Western blotting. Oxidative stress was visualized by live cell imaging and quantification of antioxidant proteins, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, platelet endothelial cell adhesion molecule 1, cyclooxygenase 2, and mitochondrial deoxyribonucleic acid by real-time polymerase chain reaction. Dissipation of the mitochondrial membrane potential was assessed with cytofluorimetric analysis using the J-Aggregate (JC-1 staining [cytofluorimetric analysis using the J-Aggregate]).

RESULTS:
Ropivacaine exposure dose-dependently induced apoptosis and an increased release of IL-6, IL-8, and PGE2 from HUVECs and TBs. Furthermore, caspase-3, nuclear factor-κB, and p38 mitogen-activated protein kinase pathways were activated, while extracellular signal–regulated kinase 1/2 and protein kinase B (Akt) were dephosphorylated. Downregulation of antioxidative proteins induced oxidative stress and upregulation of ICAM1, VCAM1, and PECAM1 possibly facilitate leukocyte transmigration. Mitochondrial effects included increased release of the proinflammatory mitochondrial DNA damage–associated molecular patterns, but no significant dissipation of the mitochondrial membrane potential. Conversely, lidocaine exhibited repression of IL-6 and IL-8 release over all time points, and early downregulation of COX2 and cell adhesion molecules, which was followed by a late overshooting reaction. Dexamethasone reduced especially inflammatory effects, but as an inducer of mitophagy, had negative long-term effects on mitochondrial function.

CONCLUSIONS:
This study suggests that ropivacaine causes cellular injury and death in HUVECs and TBs via different signaling pathways. The detrimental effects induced by ropivacaine are only partially blunted by dexamethasone. This observation strengthens the importance of inflammation in ERMF.