DOI: 10.5281/zenodo.14941962
title: Insights into the involvement of TRPA1 channels in the neuroinflammatory machinery of trigeminal neuralgia

description: This dataset comprises the findings obtained in the study aimed at investigating the putative involvement of transient receptor potential ankyrin type-1 (TRPA1) channels in the inflammatory pathways following the development of trigeminal neuralgia (TN), with a special focus on the possible modulation of glial activity after TRPA1 blockade and the cross-talk of TRPA1 with toll-like receptors 4 (TLR4) and 7 (TLR7).

A rodent model (male adult rats) of TN was established by chronic constriction injury of the infraorbital nerve and the effects of TRPA1 antagonism through ADM_12 treatment (30mg/kg, i.p.) was explored following the development of mechanical allodynia (i.e., 26 days post-surgery). The trigeminal system-related areas (trigeminal ganglion and areas containing the trigeminal nucleus caudalis) and plasma samples were used to evaluate central and peripheral inflammatory mediators (by rt-PCR and ELISA) and immunofluorescence staining of glial activation in the trigeminal nucleus caudalis.

 

- Gene expression analysis: The medulla, upper cervical spinal cord (C1-C2, CSC) and the trigeminal ganglion ipsilateral to TN injury were processed to evaluate the gene expression levels of Arginase 1, Complement 3 (C3), cluster of differentiation molecule 11b (CD11b), glial fibrillary acidic protein (GFAP), high mobility group box 1 (HMGB1), inducible nitric oxide synthase (iNOS), interleukin (IL) 10, IL-4, S100 Calcium Binding Protein A10 (S100a10), TLR4, TLR7, RNU6-6P (U6), miR-let-7b.

mRNA levels were measured by rt-PCR. All samples were assayed in triplicate and averaged; the 2−∆∆Ct method was used to evaluate the amount of mRNA normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or RNU6-6P (U6).

 

- Immunofluorescence staining: Following transcardial perfusion with 4% paraformaldehyde, the medullary segment containing the trigeminal nucleus caudalis was removed and sectioned (30 µm) on a freezing sliding microtome. Immunofluorescence staining of CD11b (microglia marker) and GFAP (astroglia marker) was assessed in the trigeminal nucleus caudalis. CD11b- or GFAP-positive cells were counted from a stack of 16 pictures (1μm-thick, 20-fold magnification) of four representative sections alongside the trigeminal nucleus caudalis. The total number of CD11b- and GFAP-positive cells of the four sections were summed and expressed per mm2. From the same sections, a qualitative analysis of glial activation state within the trigeminal nucleus caudalis was determined by using a score system ranging from 0 to 3. The final activation state was given by the average of the scores assessed for each of the four representative sections of the trigeminal nucleus caudalis.

- Protein plasma levels: tumor necrosis factor alpha (TNF-alpha), IL-1beta, IL-6, IL-10, IL-4 and HMGB1 levels were measured with commercial ELISA kits.

 

 

RESULTS:

Compared to sham-operated rats, the TN-like animals showed significantly more marked activation of microglia and astroglia in the trigeminal nucleus caudalis, with higher and lower protein plasma levels of pro-inflammatory and anti-inflammatory cytokines, respectively. Additionally, in the trigeminal-related areas, TN-like animals showed significantly higher gene expression levels of TLR4, TLR7, miR-let-7b, and high mobility group box-1. TRPA1 antagonism reverted all the observed alterations in TN-like rats in the trigeminal-related areas and plasma except glial activation in the trigeminal nucleus caudalis.