Ashley White

Introduction. Fetal Alcohol Spectrum Disorders (FASD) are a group of conditions that can occur in individuals who were exposed to alcohol prenatally. The disorder causes of a variety of physical and cognitive issues in those afflicted, including executive functioning disorders, learning deficits, physical dysmorphisms, and growth deficiencies¹. The condition is estimated to have a prevalence of 31.1 to 98.5 per 1000 children in the US¹, making it a major public health issue. While the exact mechanism behind the development of FASD is unknown, there are many pathways theorized to be involved. One of which is the TLR4-induced inflammatory pathway. TLR4, or Toll-Like Receptor 4 is a protein expressed by innate immune cells². The traditional ligand of TLR4 is lipopolysaccharide, but ethanol can also promote TLR4 activity through interaction with cell membrane lipid rafts². Studies have shown that ethanol-driven stimulation of TLR4 leads to the activation of an inflammatory process in the brain^2,4. Methods. WT and TLR4-knockout female mice were fed a diet with or without ethanol during gestation and lactation. Some dams were euthanized to obtain fetuses. Cytokine levels in the amniotic fluid and the cerebral cortices of fetuses were measured using ELISA. Microglial and neuronal markers, myelin protein, and synaptic protein levels were assessed in the cortices of WT and TLR4-knockout pups using immunohistochemical staining, western blotting, and electron microscopy. Behavioral testing of the offspring was carried out consisting of an elevated maze puzzle (to evaluate anxiety) and a passive avoidance puzzle (to evaluate memory and learning). Results. Cytokines were found to be upregulated in the amniotic fluid and cortices of the prenatally ethanol exposed (PEE) WT fetuses and offspring but not in PEE TLR4 knockout mice. Ethanol treatment significantly downregulated the levels of myelin-related proteins in the PEE WT pups compared to the levels found in PEE TLR4-knokout pups. PEE WT mice spent more time in the closed-off portions of the maze and showed shorter latencies in the passive avoidance test than their respective control group and PEE TLR4-knockkout mice, suggesting that PEE induces anxiety-related behavior and leads to learning and memory dysfunctions, respectively. Conclusions. Studies have shown that ethanol stimulated activation of TLR4 not only results in the induction of an inflammatory cascade in the brain, but that this inflammation is linked to the development of cognitive deficits^2,3. Suppressing the activity of TLR4 in the fetal brain during gestation could serve to protect the fetus from damage caused by maternal alcohol consumption.

1. May, P.A., Chambers, C.D., Kalberg, W.O., Zellner, J., Feldman, H., Buckley, D., Kopald, D., Hasken, J.M., Xu, R., Honerkamp-Smith, G., et al. (2018). Prevalence of Fetal Alcohol Spectrum Disorders in 4 US Communities. JAMA 319, 474-482.
2. Pascual M, Montesinos J, Montagud-Romero S, et al. TLR4 response mediates ethanol-induced neurodevelopment alterations in a model of fetal alcohol spectrum disorders. J Neuroinflammation. 2017;14(1):145. Published 2017 Jul 24. doi:10.1186/s12974-017-0918-2
3. Tulina NM, Brown AG, Barila GO, Elovitz MA. The Absence of TLR4 Prevents Fetal Brain Injury in the Setting of Intrauterine Inflammation. Reprod Sci. 2019;26(8):1082-1093. doi:10.1177/1933719118805859
4. Ibáñez F, Montesinos J, Ureña-Peralta JR, Guerri C, Pascual M. TLR4 participates in the transmission of ethanol-induced neuroinflammation via astrocyte-derived extracellular vesicles. J Neuroinflammation. 2019;16(1):136. Published 2019 Jul 4. doi:10.1186/s12974-019-1529-x