**4. Materials and Methods**

## *4.1. Animals*

Male Sprague-Dawley rats (Charles River, weighing 225–250 g at arrival) were used following the International Association for the Study of Pain (IASP)'s guidelines for pain research in animals [109]. Animals were housed in groups of 2 with water and food available ad libitum, and kept in a colony room (humidity: 45 ± 5%; room temperature: 21 ± 1 ◦C). Rats were kept under a reversed 12:12 h dark/light cycle (lights on at 20 h). All procedures were in accordance with the European Convention for Care and Use of Laboratory Animals, and were approved by the Ethical Committee for Animal Testing (*Ethische Commissie Dierproeven*, ECD) of the University of Antwerp (number 2017-16, approval 20/02/2017).

Rats were allowed to acclimate for 8 days to the housing conditions before the surgery; they were habituated to the behavioral test procedure daily for three days before pre-operative testing. Habituation and testing were conducted in a darkened room (light provided by a 60 W red light bulb suspended 1 m above the observation area) with a 45 dB background noise.

### *4.2. Surgery*

The IoN-CCI was performed as previously described [18,25,27]. Rats were anaesthetized with pentobarbital (60 mg/kg, intraperitoneally (i.p.)) and treated with atropine (0.1 mg/kg, i.p.). The surgery was performed under direct visual control using a Zeiss operation microscope (×10–25). The rat's head was fixed in a stereotaxic frame and a mid-line scalp incision was made, exposing the skull and nasal bone. The edge of the orbit was dissected free, and the orbital contents were deflected with a cotton-tipped wooden rod to give access to the left IoN, which was loosely ligated with two chromic catgut ligatures (5-0) (2 mm apart). The scalp incision was closed using polyester sutures (4-0; Ethicon, Johnson & Johnson, Belgium). In sham operated rats, the IoN was exposed using the same procedure, but the nerve was not ligated.
