Estradiol Treatment Enhances Behavioral and Molecular Changes Induced by Repetitive Trigeminal Activation in a Rat Model of Migraine

A migraine is a neurological condition that can cause multiple symptoms. It is up to three times more common in women than men, thus, estrogen may play an important role in the appearance attacks. Its exact pathomechanism is still unknown; however, the activation and sensitization of the trigeminal system play an essential role. We aimed to use an animal model, which would better illustrate the process of repeated episodic migraine attacks to reveal possible new mechanisms of trigeminal pain chronification. Twenty male (M) and forty ovariectomized (OVX) female adult rats were used for our experiment. Male rats were divided into two groups (M + SIF, M + IS), while female rats were divided into four groups (OVX + SIF, OVX + IS, OVX + E2 + SIF, OVX + E2 + IS); half of the female rats received capsules filled with cholesterol (OVX + SIF, OVX + IS), while the other half received a 1:1 mixture of cholesterol and 17β-estradiol (OVX + E2 + SIF, OVX + E2 + IS). The animals received synthetic interstitial fluid (SIF) (M + SIF, OVX + SIF, OVX + E2 + SIF) or inflammatory soup (IS) (M + IS, OVX + IS, OVX + E2 + IS) treatment on the dural surface through a cannula for three consecutive days each week (12 times in total). Behavior tests and immunostainings were performed. After IS application, a significant decrease was observed in the pain threshold in the M + IS (0.001 < p < 0.5), OVX + IS (0.01 < p < 0.05), and OVX + E2 + IS (0.001 < p < 0.05) groups compared to the control groups (M + SIF; OVX + SIF, OVX + E2 + SIF). The locomotor activity of the rats was lower in the IS treated groups (M + IS, 0.01 < p < 0.05; OVX + IS, p < 0.05; OVX + E2 + IS, 0.001 < p < 0.05), and these animals spent more time in the dark room (M + IS, p < 0.05; OVX + IS, 0.01 < p < 0.05; OVX + E2 + IS, 0.001 < p < 0.01). We found a significant difference between M + IS and OVX + E2 + IS groups (p < 0.05) in the behavior tests. Furthermore, IS increased the area covered by calcitonin gene-related peptide (CGRP) immunoreactive (IR) fibers (M + IS, p < 0.01; OVX + IS, p < 0.01; OVX + E2 + IS, p < 0.001) and the number of neuronal nitric oxide synthase (nNOS) IR cells (M + IS, 0.001< p < 0.05; OVX + IS, 0.01 < p < 0.05; OVX + E2 + IS, 0.001 < p < 0.05) in the caudal trigeminal nucleus (TNC). There was no difference between M + IS and OVX + IS groups; however, the area was covered by CGRP IR fibers (0.01 < p < 0.05) and the number of nNOS IR cells was significantly higher in the OVX + E2 + IS (p < 0.05) group than the other two IS- (M + IS, OVX + IS) treated animals. Overall, repeated administration of IS triggers activation and sensitization processes and develops nociceptive behavior changes. CGRP and nNOS levels increased significantly in the TNC after IS treatments, and moreover, pain thresholds and locomotor activity decreased with the development of photophobia. In our model, stable high estradiol levels proved to be pronociceptive. Thus, repeated trigeminal activation causes marked behavioral changes, which is more prominent in rats treated with estradiol, also reflected by the expression of the sensitization markers of the trigeminal system.