Short-Term Functional and Morphological Changes in the Primary Cultures of Trigeminal Ganglion Cells

Abstract

Several studies have proved that glial cells, as well as neurons, play a role in pain pathophysiology. Most of these studies have focused on the contribution of central glial cells (e.g., microglia and astrocytes) to neuropathic pain. Likewise, some works have suggested that peripheral glial cells, particularly satellite glial cells (SGCs), and the crosstalk between these cells and the sensory neurons located in the peripheral ganglia, play a role in the phenomenon that leads to pain. Nonetheless, the study of SGCs may be challenging, as the validity of studying those cells in vitro is still controversial. In this study, a research protocol was developed to examine the potential use of primary mixed neuronal–glia cell cultures obtained from the trigeminal ganglion cells (TGCs) of neonate mice (P10–P12). Primary cultures were established and analyzed at 4 h, 24 h, and 48 h. To this purpose, phase contrast microscopy, immunocytochemistry with antibodies against anti-βIII-tubulin and Sk3, scanning electron microscopy, and time-lapse photography were used. The results indicated the presence of morphological changes in the cultured SGCs obtained from the TGCs. The SGCs exhibited a close relationship with neurons. They presented a round shape in the first 4 h, and a more fusiform shape at 24 h and 48 h of culture. On the other hand, neurons changed from a round shape to a more ramified shape from 4 h to 48 h. Intriguingly, the expression of SK3, a marker of the SGCs, was high in all samples at 4 h, with some cells double-staining for SK3 and βIII-tubulin. The expression of SK3 decreased at 24 h and increased again at 48 h in vitro. These results confirm the high plasticity that the SGCs may acquire in vitro. In this scenario, the authors hypothesize that, at 4 h, a group of the analyzed cells remained undifferentiated and, therefore, were double-stained for SK3 and βIII-tubulin. After 24 h, these cells started to differentiate into SCGs, which was clearer at 48 h in the culture. Mixed neuronal–glial TGC cultures might be implemented as a platform to study the plasticity and crosstalk between primary sensory neurons and SGCs, as well as its implications in the development of chronic orofacial pain.

Presence of Small-Conductance Calcium-Activated Potassium (SK) Channels in the Central and Peripheral Nervous Systems and Their Role in Health and Disease

“…SK3 immunolabeling has also been found in early-cultured cells [49]. Our group has evaluated changes that occur in SK3 expression in cultured trigeminal ganglion cells, and found important variations [51]. It was possible to detect a high expression of SK3, with some cells double-staining with ßIII-tubulin at 4h in culture.…”

“…According to one preclinical study, SK3 is confined to the SGCs and not expressed by the neurons of the trigeminal ganglion [50]. However, this concept must be taken cautiously when interpreting the results of in vitro studies, since phenotypic changes involving SK channels can occur in cell cultures of the trigeminal ganglion [51]. However, these results shed light on the expression of SK channels in the trigeminal ganglion cells and suggest that targeting SK channels could be of great value in the treatment of orofacial pain, especially those of neuropathic origin.…”

Establishing a Mouse Model of Chlorpromazine-Induced Corneal Trigeminal Denervation

“…Primary TG cells were isolated following a previously reported protocol 20 and cultured in a neuronal basal medium supplemented with 10% fetal bovine serum (#10099-141; Thermo Fisher Scientific), 1% penicillin (# P1400; Solarbio Life Sciences, Beijing, China), 2% Gibco B-27 supplement (#17504044; Thermo Fisher Scientific), 1% non-essential amino acid solution (#N1250; Solarbio Life Sciences), and 1% HEPES (#H1095; Solarbio Life Sciences). Primary cells were fixed for immunofluorescence staining.…”

Trigeminal ganglion itself can be a viable target to manage trigeminal neuralgia

“…The TG is made up of glial cells and neurons[ 11 ]. The distinctive pseudounipolar architecture of TG neurons interjected links between the two terminals and the discharge of transmitters at both central and peripheral sites [ 12 ]. TG neurons can be categorized into two sizes: large and small.…”