Pregnancy is a unique experience in the lifetime of women. The mother’s health is directly connected to the fetus’s health. Therefore, it is essential to maintain the health of pregnant women. Since, local anesthetics are commonly used for surgical procedures in the pregnant women [31-33], it is necessary to understand the effects of maternally administered local anesthetics on the fetus. For this purpose, this study aimed to elucidate the adverse effects of maternal bupivacaine use on fetus hippocampal cell apoptosis and the possible related mechanism. To assess the activation of caspase-dependent apoptosis pathways in fetal hippocampal following maternally administered bupivacaine, we measured activation of caspase-3 and caspase-8. It is revealed that caspase-3 play a key role in apoptosis and its cleavage represents its activation [27, 34]. Caspase-3 is activated during both extrinsic and intrinsic apoptosis pathways [27, 35]. Also, caspase-8 is the initiator caspase that play a critical role in the extrinsic apoptotic signaling pathway. Our result revealed that maternal bupivacaine use could increase apoptosis-related proteins, cleaved caspase-3 and caspase- 8 expressions in the hippocampal of the fetus compared with the sham group.
A growing body of researches in the last years, both laboratory and clinical settings, reported that, although happened in rare-event situations, local anesthetic reagents, like bupivacaine, ropivacaine, lidocaine and mepivacaine, might induce severe neurological injury in both animal and humans [14, 36-38]. In this regard, Yu et al (2017) shown that in neuronal population, bupivacaine could significantly increase apoptosis and induce much severe neurotoxicity than other local anesthetics, such as mepivacaine or procaine [10]. In addition, another study showed that bupivacaine could induce neural apoptosis and neurite degeneration in DRG neurons [11]. Available evidences shows that local anesthetics have systemic absorption, and placenta does not limit the fetal transfer of maternally administered amide-linked local anesthetics, such as bupivacaine [19, 20, 39]. It is revealed that bupivacaine crosses the placenta, accumulates in this tissue and is retained in fetal tissues [18, 21, 22]. Bupivacaine enters in the fetal liver through umbilical venous blood perfused in this organ. The 3-hydroxybupivacaine can remain detectable in the fetus liver up to 4 h. This suggesting that bupivacaine can metabolize in the fetal liver [22]. The fetus eliminates the bupivacaine by diffusing it into the maternal compartment through the placental membrane. Although the majority of the bupivacaine metabolites are more polar, and it is unlikely that the placental membrane crosses these metabolites back into the maternal compartment, possibly resulting in the accumulation of metabolites in various fetal tissues. The half of the fetal circulation directly reaches the heart and brain, thus it is possible that reduced fetal ability to remove drugs can cause prolonged adverse effects on these tissues [40, 41]. Although there are no adequate and well-controlled studies about the adverse effect of maternal bupivacaine on the fetus, our results for the first time suggest that, maternally administered bupivacaine, could have an adverse effect on fetal brain and induce hippocampal cell apoptosis.
The exact mechanism by which bupivacaine induces apoptosis have not been elucidated entirely. However different studies have reported that several signaling pathways, such as PERK [12, 13], IRE1 [3], GSK3 [14], MAPK [15, 16] and Akt [4, 12], might be responsible for bupivacaine- induced apoptosis. In exploring the signaling mechanism; we focused on Akt, which is a well-known anti-apoptosis molecule. Our results showed that maternal bupivacaine use markedly decreases the phosphorylation levels of Akt. A number of studies have demonstrated that Akt, a key kinase downstream of the PI3-kinase, plays a crucial role in cell survival and death pathway of neurons [12, 29, 42, 43]. Recent studies have confirmed that Akt-signaling pathway involves in the bupivacaine-induced apoptosis in adults [44, 45]. In this regard, Fan et al (2016) reported that bupivacaine-induced neurotoxicity in SH-SY5Y cells is mediated thorough inactivating Akt signaling pathway [1]. In consistent with previous studies, we observed that bupivacaine decreased the phosphorylation levels of Akt in the fetal hippocampus, which was concurrent with an increment of apoptotic markers.