All animals were purchased from the Experimental Animal Center of Central South University. The experimental procedures were approved by the Animal Management and Use Committee of the Third Xiangya Hospital of Central South University, and was in compliance with the Guide for the Care and Use of Laboratory Animals published by the Chinese National Institute of Health. Before any experiment, adult male SD rats (200-220 g, a total of 30) were were raised and maintained under standard laboratory conditions, and allowed to acclimate in the cage for 12/12 hours under light-dark cycle, and they were able to eat and drink at any time.
Construction of a trigeminal pathological pain model
The rats tested were subjected to relevant adaptive training 2 weeks before the start of the experiment, and rats with stable mood expression against VonFrey hair were selected for modeling. The animals were fasted for 12 h before the experiment, but had access to water ad libitum. They were anesthetized with an intraperitoneal injection of Pentobarbital (3%, 30mg/kg). The unilateral infraorbital nerve was loosely ligated as described by Kernisant(7) to establish a trigeminal pathological pain model. The infraorbital nerve was loosely ligated with absorbable chromium gut. The ligation standard was that the diameter of the nerve was slightly thinned after ligation under the operating microscope, but the conduction could not be completely blocked, and the blood circulation of the epicardium remained unobstructed.
Thirty SD rats were randomly divided into control group, sham group, ION-CCI group, NS group (ION-CCI + normal saline) and RuR group (ION-CCI + ruthenium red), with 6 rats in each group. ION-CCI was induced by ligation of the unilateral infraorbital nerve with chromium gut loosening to establish an animal model of TN. The rats in the control group were not treated, and the surgical procedure of rats in sham group were the same as the ION-CCI group except that the infraorbital nerve was not ligated, that is, only the nerves were exposed and not ligated. On the 9th day after surgery, rats in the RuR group were intraperitoneally injected with CALHM1 inhibitor ruthenium red 0.5mg/(kg•rat•day), while the rats in the NS group was injected with an equal volume of physiological saline. In control group, sham group and ION-CCI group, mice were euthanized by three times the anesthetic dose of pentobarbital after behavioral tests. In NS group and RuR group, mice were anesthetized by Pentobarbital (3%, 30mg/kg) and euthanized by obtaining brain tissue.
Pain behavior test
Determination of mechanical pain threshold: All experimental rats should be acclimated to the environment for two weeks in advance, and pain behavioral measurements were performed 1 day before surgery (ION surgery or sham surgery) and 1, 3, 5, 7, 9, 11, 14 days after treatment. According to Vos BP et al.(8), the Von Frey filament is placed near the center of the whisker pad so that the filament is slightly curved. Stimulation of each intensity was stimulated at least 3 times from the minimum stimulation intensity, and the shortest interval between each 2 stimulations was 30 seconds, which is considered a positive reaction if one or more of the following behaviors occur: (1) Rapid withdrawal response in rats; (2) Escape or attack response; (3) The asymmetrical facial trimming behavior is characterized by more than 3 consecutive strokes of the stimulated facial area. The minimum value of the filament strength that causes a positive reaction in the rat is the mechanical pain threshold of the surgical side whisker pad. Measurements were not performed in the order of grouping, and the order of placement of rats was disrupted, and random measurements were made by those who did not understand the experimental group of rats to reduce experimental errors.
Video recording of the number of scratches: According to Spradley(9), rats were placed in a colorless transparent glass lattice that did not interfere with rats' random movement. Rats were used to adapt to the environment of the lattice in advance, and they were obtained to move freely for about an hour before they began video recording. The video observation lasted about 30 minutes. The observers were not informed of the grouping before the experiment, and the number of scratches of each group of rats at the corresponding time points (1 day before surgery and 1, 3, 5, 7, 9, 11, and 14 days after surgery) was recorded. Keep quiet during the recording process so as not to scare the rats into affecting the experimental results.
Western blotting analysis
On the 15th day after operation, the trigeminal nucleus of the rat trigeminal spinal nucleus was removed under low temperature and aseptically under the direction of the brain stereotaxic instrument. The lysate containing the protease inhibitor was added, and the tissue was homogenized and placed in 4 °C. After centrifugation at 12,000 rpm for 15 minutes in a centrifuge, the supernatant was taken and the protein content of the sample was determined by (Bicinchoninic acid) BCA quantification. The protein was then denatured by adding a loading buffer and drying at 100 ° C for 5 min. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransformation, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane and blocked for 2 hours with 5% skim milk. Rabbit anti-CALHMl primary antibody (1:1 000) diluted with 5% TBST was added and left overnight at 4 °C. After washing the membrane, the mouse secondary antibody (1:5000) was separately added, and the membrane was then incubated at 37℃ for 1 h, and then subjected to ECL coloration and exposure. Semi-quantitative analysis was performed using β-actin as an internal control.
All data were expressed as mean ± standard deviation, and the results of western blot were analyzed using Image J for gray scale analysis. Two-Way ANOVA was used to compare the mechanical pain threshold and video behavioral results of each group of rats, and comparison between groups was performed at the same time point. The difference of gray value of each group was analyzed by one-way ANOVA and then compared by Tukey method. The difference was statistically significant (P< 0.05). The data were analyzed and plotted by Graphpad Prism 6 software.