Insights into the involvement of TRPA1 channels in the neuroinflammatory machinery of trigeminal neuralgia

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.