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International Journal of Bioprinting Pregabalin impact on 3D neuronal models

neurons. These neurons are mainly responsible for both comprehensive coverage and precision. Our study
the complex brain activity that underlies cognition, employed a cutting-edge 3D bioprinter, as described
perception, and voluntary movements. More than 12 types by Susapto et al., to fabricate 3D structures composed
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of cortical neurons can be categorized based on whether of cortical neurons embedded within IIZK-based
they stimulate or block neural activity in the brain. The bioinks. IIZK, an ultrashort self-assembling peptide
cortical neurons are the primary functional cell type in the hydrogel, demonstrated excellent biocompatibility and
cerebral cortex of the brain. rheological properties suitable for supporting neurons
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Within the developing brain, Tbr1, which is a neuron- and other cell types, as reported in recent studies. 29,30
specific T-box transcription factor, plays a crucial role Specifically, we aimed to assess the efficiency of high-
in determining the regional and laminar identity of throughput 3D bioprinting technology, which previously
neocortical regions, including layer 6. Additionally, demonstrated its efficacy in cancer research, for its
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Tbr1 is involved in the regulation of gene expression for application in neuropharmacology. Our study focused
proteins such as reelin and the GluN2B component of the on the use of 3D bioprinting for the high-throughput
N-methyl-D-aspartate (NMDA) receptor. These proteins screening of cortical neurons treated with pregabalin.
are key in the regulation of brain development and This innovative approach facilitates the rapid assessment
functionality. Furthermore, Tbr1’s expression is increased of pregabalin’s impact on neuronal morphology and
in response to neuronal activity, in conjunction with function and highlights the potential of high-throughput
calcium/calmodulin-dependent serine kinase (CASK) (a 3D bioprinting in revolutionizing drug discovery and
synaptic PDZ protein capable of nuclear translocation) evaluation in neuropharmacology. High-throughput 3D
and CASK-interacting nucleosome assembly protein bioprinting enables efficient and extensive screenings,
(CINAP) (which plays a role in nucleosome assembly). 21,22 thereby accelerating the pace of neuropharmacological
Tbr1 is a transcription factor, with significant expression research and potentially uncovering novel therapeutic
levels in early-born glutamatergic cortical neurons. These avenues for neurological disorders. This methodological
putative transcription factors govern the regional and advancement promises to enhance our understanding of
laminar identities of postmitotic cortical neurons in a drug–neuron interactions significantly, paving the way for
coordinated manner. 23,24 Tbr1 is expressed not only in the development of more effective treatments for a myriad
layer 6 but also in the pre-plate neurons, which represent of neurological conditions.
the early mature neurons in the cortex, which is why we This study aims to enhance our understanding of
focused on this population in our study. Our objective pregabalin’s effects on neuronal behavior within a more
was to explore the impact of pregabalin exposure on the naturalistic biological environment. Additionally, it
initial development stages of isolated mouse embryonic seeks to provide vital information for enhancing safety
cortical neurons (ECNs) to assess if pregabalin exposure considerations during pregnancy upon administration
influences the morphogenesis and differentiation of of the drug. We also aim to demonstrate the efficacy
cortical forebrain neurons. Specifically, we investigated the of high-throughput 3D bioprinting technologies in
effects of pregabalin on forebrain cortical neuron survival, neuropharmacological applications.
adenosine triphosphate (ATP) release, morphogenesis,
gene expression, and neuronal activity. To closely replicate 2. Materials and methods
the characteristics of natural brain tissue, we established 2.1. Isolation of primary mouse embryonic
a 3D cell culture model using tetrameric self-assembling cortical neurons
peptide matrices. Recently published results have Mouse embryonic cortical neurons (ECNs) were isolated
demonstrated the superior performance of 3D culture from E12.5 cortices to examine their properties. Swiss
systems compared to conventional 2D approaches. mice were bred to obtain embryos, from which ECNs were
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These peptides can form intricate nanofibrous networks extracted. No control group was included, as we aimed to
resembling the structural composition of the extracellular study the inherent characteristics of ECNs after exposure
matrix (ECM), specifically mimicking collagen fibers. 26,27 to pregabalin in vitro, providing foundational baseline
The peptide scaffolds were selected based on the promising data. The mice were kept at room temperature (20 ± 2°C),
outcomes of earlier research, where tetrameric self- humidity of 50–70%, and a daylight cycle of 12 h dark
assembling peptides demonstrated their effectiveness as a and 12 h light. The mice had ad libitum access to fresh
platform for creating robust 3D neuronal models. 28,29 drinking water and food (a conventional rodent diet). The
The emergence of high-throughput 3D bioprinting animal experiments were carried out in compliance with
technology offers unprecedented opportunities to explore the international norms for the use of animals in research.
the intricate dynamics of cell–drug interactions with All experimental protocols were approved by the ethics

Volume 10 Issue 4 (2024) 406 doi: 10.36922/ijb.3010