Transmembrane protein 100 is expressed in neurons and glia of dorsal root ganglia and is reduced after painful nerve injury

Abstract

Supplemental Digital Content is Available in the Text.

Piezo2 mechanosensitive ion channel is located to sensory neurons and non-neuronal cells in rat peripheral sensory pathway: implications in pain

“…All IB4- and CGRP-positive neurons which consist of 45% and 59% of Piezo2-positive neurons, respectively, were Piezo2 immunopositive, indicating that Piezo2 is highly expressed by unmyelinated (C-type) and thinly myelinated (Aδ-type) PSNs that convey the thermal and mechanoreceptive nociceptive signals generated at peripheral nerve terminals to neurons in lamina I-II of the spinal cord [17]. Nociceptive neurons in adult rat DRG can be double positive for both IB4 and CGRP (30-40%) [80], suggesting that there is a substantial proportion of small Piezo2-IR neurons positive for both IB4/CGRP. Colabeling of Piezo2 with NKA1 α revealed enriched Piezo2 profiles in the PSN plasma membrane, especially those of larger diameter neurons.…”

“…For quantitative comparison, we normalized Piezo2-IR intensity on the side ipsilateral to CFA by using the contralateral side in the same section as the control [14, 57], assuring that all IHC preparation and image capture parameters are identical in the same cross-section. To validate the use of general IR intensity of the DH as a quantitative indicator of protein expression in sensory neurons, we compared IB4 and CGRP staining in the DH from SNI rats, in which quantitatively decreased IB4 and CGRP immunoreactive intensity in sensory neurons ipsilateral to the SNI injury is expected [37, 80]. As shown in Suppl.…”

“…Intensity correlation analysis (ICA) was performed to determine colocalization of Piezo2 with neuronal plasma membrane (PM) marker sodium/potassium ATPase 1 alpha (NKA1a) and SGC marker glial fibrillary acidic protein (GFAP), as previously described using an ImageJ 1.46r software plugin colocalization analysis module (http://imagej.nih.gov/ij) [35, 58, 80]. In brief, fluorescence intensity was quantified in matched region of interests (the green and red colors varied in close synchrony) for each pair of images.…”

“…Statistical analysis was performed with GraphPad PRISM 8 (GraphPad Software, San Diego, CA). Significances of ICQs of Piezo2 immunocolocalization with NKA1 α and GFAP were analyzed by means of the normal approximation of the nonparametric Wilcoxon rank test, as described previously [35, 80]. Mechanical allodynia and hyperalgesia were compared between groups by Student’s t test for von Frey and by Mann–Whitney test for Pin.…”

“…For western blot experiments, L4/L5 DRG from CFA-and saline-injected rats, after cardiac perfusion with cold PBS, were dissected, snap frozen in liquid nitrogen, and stored at −80°C for extraction of protein. [78,80]: In brief, the L4/5 DRG were rapidly harvested from the isoflurane anesthetized naïve rats and were incubated in 0.01% blendzyme 2 (Roche Diagnostics, Madison, WI) for 30 min, followed by incubation in 0.25% trypsin and 0.125% DNase for 30 min; both dissolved in Dulbecco's modified Eagle's medium/F12 (DMEM/F12) with glutaMAX (ThermoFisher). After exposure to 0.1% trypsin inhibitor and centrifugation, the pellet was gently triturated and dissociated cells cultured in Neural basal media A (ThermoFisher) plus 0.5 µM glutamine at 37°C in humidified 95% air and 5% CO2.…”