1000: Increased amylase activity in the amniotic fluid of fetal rats with retinoic acid induced myelomeningocele

Abstract

Neurologic injury in myelomeningocele (MMC) occurs via a two-hit process: failed neural tube closure followed by neurodegeneration in utero. Meconium in the amniotic fluid causes neurotoxicity in rat models of MMC and contains digestive enzymes from the pancreas. The objective of this study is to measure the enzyme activity of amylase in the amniotic fluid of rats affected by retinoic acid induced MMC. We hypothesize that amylase activity will be present at increasing levels with advancing gestation and will be higher in the MMC group compared to a control population at all gestational ages. Timed pregnant Sprague Dawley rats were gavage fed all-trans Retinoic Acid (60 mg/kg) in olive oil on gestational day E10 to induce MMC defect (model previously described). Control rats received olive oil. Amniotic fluid was collected on embryonic days E15, E17, E19 and E21. MMC defect was confirmed visually. Amylase activity was assessed at each gestational age using the EnzChek Ultra Amylase Activity Assay Kit (Invitrogen, Carlsbad CA). Amniotic fluid samples were plated in triplicate on a 96 well microplate (N=8 per group). Amylase activity was detected via fluorescence at Ex 505nm/Em 512nm and read with a microplate reader (Glomax-Multi Promega, Madison, WI). Levels were compared between MMC and control groups at each gestational age using Wilcoxon Rank Sum and Kruskal-Wallis tests. Amylase activity for embryonic days E15, E17, E19 and E21 was determined for MMC and control pups. Amniotic fluid amylase activity increased significantly from E15 to E21 in both control (p=3.0 x 10-5) and MMC (p=1.5 x 10-5) groups. Amylase activity was significantly increased in MMC pups compared to controls on E19 (247,792.8 vs. 106,263.6; p=0.0019) and E21 (772,645.8 vs. 481,975.3; p=0.021); no difference was detected on E15 (36,646.8 vs. 40,179.3; p=0.645) or E17 (121,617.5 vs. 71,750; p=1.000). (Graph 1). Amylase activity level is higher in rats with retinoic acid induced MMC compared to controls with advancing gestational age. The higher activity of this digestive enzyme in fetuses with MMC may be a contributor to neural tube damage in utero. Future research should focus on amylase and other digestive enzymes as potential targets of in utero therapy in patients who are not candidates for antenatal MMC repair.