Bio evf3

As previously described [24 (link)], head withdrawal thresholds (HWTs) were tested by applying mechanical stimulation to the ipsilateral or contralateral masseter muscle using a modified testing probe of an electronic von-Frey anesthesiometer (BIO-EVF3, Bioseb, Vitrolles, France). Prior to EOI placement or viral vector injection surgery, rats were placed on the palm of the experimenter's hand and acclimatized to the test environment, without any restraint equipment or methods, to allow them to retract their head freely (Fig. S1). The force applied to the orofacial region was recorded five times to elicit a head withdrawal response, and the average of the five values was calculated as the HWT for that measurement. The average of the HWTs measured on each day of the following 3 days was calculated as the baseline HWT. HWTs measurements were performed in a blinded fashion by the same experimenter.

Mechanical sensitivity in the masseter was tested as the head withdrawal threshold using a modified electronic von-Frey aesthesiometer (BIO-EVF3, Bioseb, Vitrolles, France) as previously reported [13 (link)]. Briefly, rats were acclimated to the testing environment for 30 min for three consecutive days before baseline evaluation. The rat was free to withdraw its head from the progressively increasing mechanical stimulus over the belly region of the masseter. The applied force in grams was automatically recorded when the head was withdrawn. The head withdrawal thresholds were averaged based on five measurements per masseter. The baseline mechanical threshold was determined over three continuous days. All data reported in the behavioral experiment were evaluated by a trained tester with blind method.

Cutaneous allodynia is a typical symptom of migraine, with a crucial role in directing optimal treatment for migraine attacks (Landy et al., 2004 (link)). Cutaneous allodynia was measured by von Frey monofilaments by assessing the mechanical thresholds in the periorbital region of a rat. In brief, rats were placed in a plastic box to acclimate the situation before testing. Facial allodynia on the periorbital region of a rat was assessed by Electronic Von Frey Filaments (BIO-EVF3, BIOSEB, Vitrolles, France). The force values ranged from 0 to 500 g. A positive response was defined as a sharp retraction of the head or scratching the face with the forepaw (Wang S. et al., 2018 (link)). The test was repeated three times with an interval of at least 20 s, and the average value was calculated as the final result.