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International Journal of Bioprinting Pregabalin impact on 3D neuronal models

Figure 2. Viability and adenosine triphosphate (ATP) release in pregabalin-treated and control cultures. (A) Viability and (B) ATP release in cultures
treated with pregabalin and control cultures. Data are presented as the mean ± standard error of the mean (SEM); n = 7 for viability and n = 3 biological
replicates for ATP release. Statistical analyses were performed using a t-test. * p < 0.05.

production, which is considered a substantial alteration. 3.4. Pregabalin induces upregulation or
Therefore, it can be concluded that pregabalin disrupts downregulation of important cortical-related genes
the mitochondrial electron transport pathway, leading to in embryonic cortical neurons
metabolic failure (Figure 2B). Genome-wide association studies (GWAS) have evolved
into an effective technique to identify the genes responsible
3.2. Pregabalin does not alter the morphogenesis of for variations in a wide range of human traits and
Tbr1-positive embryonic cortical neurons disorders. As a result of numerous GWAS, genes and
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The effects of pregabalin on the morphogenesis of Tbr1- biological pathways that are particularly active at different
positive ECNs were evaluated using labeled cultures. stages of prenatal brain development have been identified.
37
Figure 3A and B displays non-significant morphological
changes in the lengths and dominant lengths of neurites, These genes have earlier been linked to intellectual
38,39
respectively. Figure 3C and D indicates no significant disability and developmental delays. Modern genomics
variation in the number of branches or neurites between suggests that changes in gene regulation are the main
pregabalin-treated and control cultures. Moreover, factor driving the evolution of the human brain. To fully
labeled cultures from both groups revealed that there comprehend the biological role of these largely noncoding,
were no notable differences in the morphology of Tbr1- tissue-specific gene regulatory elements, it is necessary
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positive ECNs (Figure 3E–Lʹ). These findings suggest that to understand their biological functions. Here, we
pregabalin exposure, at a therapeutic dose, does not affect studied the expression of key genes involved in neuronal
the morphogenesis of Tbr1-positive ECNs. differentiation and development. The expression of Emx2,
Lhx6, and Nkx2.1 in ECNs did not change significantly
3.3. Effect of pregabalin on the morphogenesis of as a result of pregabalin exposure (Figure 5A, C, and D,
Tbr1-negative embryonic cortical neurons respectively). Although Gsx2 was downregulated in the
The effects of pregabalin on the morphogenesis of Tbr1- pregabalin-treated culture, this shift is not statistically
negative ECNs were evaluated in labeled cultures to significant (Figure 5B). However, there was a considerable
determine whether pregabalin has any specific effects shift in the regulated expression of the genes encoding
on the morphogenesis of the restricted forebrain ECN Dlx2, Olig2, NhIh2, Otp, Zic1, and Gad67 (Figure 5E–J,
subpopulation. There was no significant difference respectively). In pregabalin-treated cultures, Olig2 and
observed in terms of overall neurite length, length of the Zic1 were significantly upregulated, whereas Dlx2, NhIh2,
dominant neurite, number of branches, and total neurite Otp, and Gad67 were significantly downregulated.
count between control groups and those treated with
pregabalin (Figure 4A–D). Furthermore, analysis of the 3.5. Microelectrode array recordings of
labeled cultures from both groups revealed that there were spontaneous activity in 2D- and 3D-cultured
no prominent differences in the morphology of Tbr1- primary cortical neurons
positive ECNs (Figure 4E–Lʹ). Based on these findings, The dynamic intricacies of neural activity were studied in
pregabalin, at a therapeutic dose, does not alter the both 2D- and 3D-cultured primary cortical neurons. We
morphogenesis of Tbr1-negative ECNs in vitro. aimed to elucidate the effects of pregabalin on the electrical

Volume 10 Issue 4 (2024) 411 doi: 10.36922/ijb.3010