2.1 Experimental animal and ethical review
The present study included 72 adult female Sprague-Dawley rats (age: 8 weeks; Koatech, Pyeongtaek, Korea), each weighing 200-250 g on arrival. Animals were allowed ad libitum access to fresh food and water and maintained under a 12-hour light/dark cycle at a humidity of 50-60%. We used female animals because it has been well established that trigeminal nerve-associated disorders are more prevalent among females than among males, and that CGRP expression is significantly higher in females than in males (13). All animal tests were conducted in a randomized, double-blind, controlled manner. The animals were randomly divided into a TN group (n = 32), a sham group (n = 32) and a control group (n=8). The timeline of the experimental protocol is shown in Figure 1. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Chungbuk National University in Korea. All animal experiments were performed during the light period at the Laboratory Animal Research Center of Chungbuk National University.
2.2 Generation of trigeminal neuralgia model
Ligation of the infraorbital nerve (ION) was performed in 32 animals in accordance with the procedure described by Melanie et al., 2008 (15). Animals were anesthetized with an intraperitoneal (i.p.) injection of a mixture of 15 mg/kg Zoletil (Zoletil50®, Virbac Laboratories, Carros, France) and 9 mg/kg Rompun (Rompun®, Bayer, Seoul, South Korea) in saline, following which they were mounted onto the surgical field in a prone position. The skin above the eye was then shaved, and the animals were placed in a stereotaxic frame. Ophthalmic ointment was applied to the cornea to prevent damage associated with drying of the eyes. A skin incision of approximately 7 mm in length was made along the curve of the frontal bone in the anterior-posterior direction, 2 mm above the left eye. Moving laterally, the fascia and muscle were gently separated from the bone using a periosteal elevator. The ION could be observed on the maxillary bone following retraction of the eye. After revealing the ION, we prepared for ligature placement by gently freeing approximately 8 mm of the ION from the surrounding connective tissue. The ION was stretched slightly using a blunt needle with a curved head for ligature placement. The two ligatures were gently placed 3-4 mm apart, following which they were tightened until the ION was barely constricted. Finally, the incision above the eye was sutured with silk (3-0). Sham animals (n=32) underwent identical procedures, with the exception of ligature placement. After surgery, the rats were given fresh pelleted food and water ad libitum and checked for survival every day for at least 1 week.
2.3 Injection of optogenetic viral vector
Animals of the TN group were randomly divided into two groups. Sixteen rats were subjected to optogenetic viral vector (AAV2-CaMKII-hChR2-EYFP) injection contralateral to the ION, while the remaining 16 were subjected to null virus (AAV2- CaMKII-EYFP) injection. Vectors were intracranially injected into layer V of M1 (AP -1 mm, ML 1.5mm, DV -1.5mm; (16-18)) under general anesthesia. Animals of the sham group were also divided into two groups for injection of either the optogenetic virus or null virus. We injected an adeno-associated virus carrying the ChR2-EYFP fusion protein under the control of an excitatory neuron-specific CaMKII promoter. Prior to virus injection, animals were anesthesized via an intraperitoneal injection of a mixture of 15 mg/kg tiletamine/zolazepam (Zoletil50®, Virbac Laboratories, Carros, France) and 9 mg/kg xylazine (Rompun®, Bayer, Seoul, Seoul, South Korea). Then, 2 µl of virus was injected at a rate of 0.3 µl/min using a Hamilton syringe and an automatic micro-syringe pump. After injection, the needle was kept in the same place for 5 min to ensure virus absorption, following which it was slowly retracted.
2.4 Behavioral testing
All behavior tests were conducted 1 day before (baseline) and every 7 days after model making. The behavior tests were performed in the following sequence:
2.4.1 Air-puff test
Animals were placed in a Plexiglas cage (20 x 20 x 14 cm) with a grid floor under quiet condition. Rats were habituated to the environment for at least 30 minutes. Continuous puffs of air (starting from 5 psi, duration: 4 seconds) were delivered to the ipsilateral side to the ION of the face at intervals of 10 seconds. Air puffs were delivered through a narrow tubing tip placed approximately 1 cm from the face at an angle of 90º. The psi was increased over trials and during administration of the air puff, we measured and evaluated the psi level of air puff at which the presence of aggressive behaviors such as biting or turning the head were exhibited by the animal. If the animal exhibited no response to a stimulus of at least 40 psi, the test was ended, and the animal was given a break for a few minutes. Mechanical thresholds were evaluated and represented >50% of the overall responses (2).
2.4.2 Mechanical allodynia test
Orofacial sensitivity to mechanical stimulation was assessed using von Frey filaments. Animals were first acclimated in a Plexiglas cage for one hour. During this time, von Frey filaments were brought near the animals every 5 minutes for habituation. Animals were allowed to explore the filaments for a few seconds before they were removed. Stimuli were administered after the animals had become accustomed to the filament when placed near the center of the vibrissal pad within the territory of ipsilateral ION. The intensity of stimulation was gradually increased. The lowest force (in grams) required to elicit reactions such as immediate withdrawal, attacking the filament by biting or grabbling, escape behaviors, or asymmetric stroking of the face was recorded as the response threshold force (19).
2.4.3 Facial cold hyperalgesia test
Animals were placed in the same Plexiglas cage described above. For this test, a few drops of acetone were placed on the vibrissal pad ipsilateral to the ION using a glass syringe, following which we counted the number of scratching/rubbing behaviors over the next 2 min. Body parts other than the face were excluded for this assessment (2).
2.5 Optic fiber implantation
Four weeks after optogenetic virus inoculation, each rat was positioned in a stereotactic frame following induction of anesthesia. An optic fiber was implanted into the skull contralateral to the ION (AP: -1 mm, ML: 1.5 mm) for the transmission of laser pulses to layer V of M1. Optic fibers (200 µm core, 230 µm outer diameter, numerical aperture of 0.48, hard polymer cladding type, Doric Lenses; Québec City, Québec, Canada) were cut to a length of 1.4 mm to optimize targeting of M1. Dental cement was used to fix the fiber firmly in place (Ortho-ject Pound Package, Lang Dental, USA).
2.6 Selective knock-down of α-CGRP in trigeminal ganglion
Before performing the behavioral tests and extracellular recording with optic stimulation, animals in each group (TN-Opto, TN-Null, Sham-Opto, and Sham-Null) were again divided into two sub-groups of 8 animals each. Animals in one group received an injection of α-CGRP (α-CGRP (8-37) (mouse, rat) trifluoroacetate salt, 1 mg/ml, BACHEM) as a CGRP receptor antagonist, while animals in the other group received an injection of PBS. Animals of CGRP group received an intravenous injection of 10 µl of α-CGRP 10 minutes prior to the behavior test. In case of extracellular recording, α-CGRP was injected directly into the ipsilateral trigeminal ganglion (AP: -3.5 mm, ML: -3.6 mm, DV: -12 mm) (20) and the recording was performed after that. α-CGRP was injected at a rate of 0.3 µl/min using a Hamilton syringe and an automatic micro-syringe pump. After injection, the needle was kept in the same place for 5 minutes to ensure absorption of CGRP, following which it was slowly retracted.
2.7 Optical stimulation
We used a laser power supply with a wavelength of 473 nm (ADR-700D, Shanghai, China) and a waveform generator (Keysight 33511b-CFG001, Keysight, Santa Rosa, CA, USA) to regulate the waveform and pulse width of the laser (Figure 2). The laser’s intensity was set to 10 mW, the pulse width was set to 4 ms, the pulses were set to 20Hz and the duration of stimulation was 5 minutes (21). Behavioral differences were examined in animals inoculated with the optogenetic virus (pre, stim, post) to determine the effects of optical neuromodulation.
2.8 In vivo extracellular recording
Six weeks after ligation of the ION, rats were anesthetized with 15 mg/kg tiletamine/zolazepam and 9 mg/kg xylazine to prepare for extracellular recordings. Extracellular recordings were obtained from the VPM (AP -3.5 mm, ML 2.8 mm, DV -6 mm) (2) using a single electrode. Following a 20-min resting condition, we recorded thalamic neuronal spikes and firing rate during the pre-stimulation, stimulation (blue: 473 nm), and post-stimulation states in vivo. A glass-insulated carbon fiber microelectrode (Cat. No.: E1011-20, Carbostar-1, Kation Scientific, LLC, MN 55414 USA) was used for recording in the thalamus. Recordings were obtained for 5 min during each stage, with a 5-min gap between stages. We chose well-isolated clusters and recorded neuronal signals using a Digital Lynx SX (Neuralynx, Bozeman, USA) data-acquisition system along with Cheetah software. We digitized and bandpass filtered at 40 kHz and 1 Hz - 5 kHz, respectively. We sorted offline using Spike Sorter 3D (Neuralynx Inc., Montana, USA). Neuronal discharge was evaluated in the contralateral VPM of lesioned and sham animals. The rate histograms (spikes/s) of lesioned animals under different optical conditions were analyzed using Neuroexplorer (Neuralynx Inc., Montana, USA).
2.9 Histological examinations
The rats were deeply anesthetized and transcardially perfused with PBS followed by 4% paraformaldehyde. The brains and trigeminal ganglion were extracted and fixed overnight in the same post fixed solution, followed which they were dehydrated in 30% sucrose solution.
We embedded brains and trigeminal ganglions in optimal cutting temperature (O.C.T.) compound (Tissue Tek®- Sakura, USA), following which they were cryopreserved with liquid nitrogen and isopentane at -79°C. A cryostat (Thermo Scientific, Waltham, MA, USA) was used to cut coronal sections of the brain (20 µm) and trigeminal ganglion (10 µm). The brain sections were incubated with DAPI and mounted with coverslips for examination under fluorescence microscope. To observe the action of α-CGRP receptor antagonist in trigeminal ganglion, the animals were sacrificed immediately after the extracellular recording and the trigeminal ganglion was collected. Trigeminal ganglion sections were immunostained with anti-CGRP antibody (1:200, ab36001, Abcam), following which they were incubated in serum block solution for 1 hour and with anti-αCGRP antibody overnight. The corresponding secondary antibody was applied prior to staining with DAB (Vector Laboratory, California, USA). Nuclei were counterstained with hematoxylin. Finally, the sections were dehydrated and mounted with coverslips and examined under a microscope.
2.10 Analysis of the bursting and firing rates
Activity in thalamic neurons was divided into burst rates (bursts/s) and overall firing rates (spikes/s) in each optical stimulation condition using NeuroExplorer software (Neuralynx Inc.). Activity was assessed for 5 min in each state: pre-stimulation, stimulation, and post-stimulation. We defined burst rates as a group of at least three spikes with a maximum interval of 4 ms between spikes, with an interval of 100 ms between bursts. We selected similar inter-spike interval histograms for comparison among the groups.
2.11 Statistical analysis
Data were analyzed using GraphPad Prism (GraphPad Software version 8.4.2, Inc., San Diego, CA, USA) and represented as the mean ± standard deviation (SD). We performed either an unpaired t-test, two-way analysis of variance (ANOVA) with Tukey’s post hoc test, or a repeated-measures ANOVA depending on the conditions of the experiment. Behavioral tests were assessed based on the mean values for each of the three optical states. Unpaired t-tests were used to compare firing rates between TN and sham-operated animals. All the statistical data were measured as significant at p<0.005.