Modulation of glia activation by TRPA1 antagonism in a pre-clinical model of migraine

This dataset comprises the findings obtained in the study aimed at investigating the relationship between TRPA1 channels and glia activation in the modulation of trigeminal hyperalgesia in preclinical models of migraine.

In vivo assessments performed in male Sprague-Dawley rats four hours after treatment with nitroglycerin (10mg/kg, i.p.) or its vehicle in the acute model, and 24h after the last treatment with nitroglycerin (5mg/kg, i.p.) or its vehicle in the chronic model (injections every other day over a 9-day period for a total of five injections):

- evaluation of trigeminal hyperalgesia in the orofacial formalin test: the face rubbing was measured counting the seconds the animal spent grooming the injected area (upper lip, lateral to the nose) with the ipsilateral forepaw or hindpaw 0–3 min (Phase I) or 12–45 min (Phase II) after formalin injection (50 µl, s.c.). The observation time was divided into 15 blocks of 3 min each.

Ex vivo assessments performed on samples collected at the end of the behavioral test:

- Microglia and astroglia activation was evaluated the trigeminal nucleus caudalis by means of immunofluorescence staining. Specifically, the medullary segment containing the trigeminal nucleus caudalis (between +1 and -5 mm from the obex) was removed and cut transversely at 30 µm thickness on a freezing sliding microtome. The anti-cluster of differentiation molecule 11b (anti-CD11b, 1:300) primary antibody (to detect microglia) or rabbit anti-glial fibrillary acidic protein (anti-GFAP, 1:500) primary antibody (to label astrocytes) were used. Cell count for microglia and astroglia was assessed by counting CD11b- or GFAP-positive cells, respectively, from a stack of 16 pictures (1μm-thick, 20x magnification) taken from 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. Glial activation state was rated on a scale ranging from score 0 to 3. The final activation state was calculated as the average of the scores assessed for each of the four representative sections of the trigeminal nucleus caudalis.

- Satellite glial cells activation (anti-GFAP, 1:600), calcitonin gene-related peptide (CGRP) immunoreactivity (anti-CGRP, 1:500) and macrophage infiltration (anti-Iba1, 1:500) were evaluated in trigeminal ganglia using immunofluorescence staining. Specifically, trigeminal ganglia were removed and sectioned longitudinally at 20 µm thickness using a cryostat.  In the trigeminal ganglia, the number of Iba1+ cells/area and the % of GFAP-encircled neurons and of CGRP+ neurons were assessed in whole TG sections acquired at 20X magnification.

- Gene expression analysis: mRNA expression levels of (TNF-alpha), interleukin (IL) -1beta, IL-6, IL-10, inducible nitric oxide synthase (iNOS), GFAP, CD11b and CGRP in medulla oblongata and trigeminal ganglia. mRNA levels were measured by rt-PCR. All samples were assayed in triplicate and gene expression levels were calculated according to 2−∆∆Ct = 2− (∆Ct gene − ∆Ct housekeeping gene) formula by using Ct (cycle threshold) values.

Results - In nitroglycerin-treated rats, ADM_12 showed an anti-hyperalgesic effect in both the acute and chronic models. ADM_12 was able to counteract the acute and chronic nitroglycerin-induced changes in cytokines’ gene expression in both the areas of interest. In the acute nitroglycerin model, we reported a significant increase in microglial and astroglial activation in the trigeminalis nucleus caudalis area, which was abolished by ADM_12 treatment. In the chronic model, we detected an activation of satellite glial cells in the trigeminal ganglia in nitroglycerin-treated rats; this was inhibited by ADM_12. This latter also prevented the increase in CGRP gene expression levels in the trigeminal ganglia of nitroglycerin-treated animals.