Critical role of GRP receptor–expressing neurons in the spinal transmission of imiquimod‐induced psoriatic itch

Abstract Aim Ample evidence indicates that gastrin‐releasing peptide receptor (GRPR)–expressing neurons play a critical role in the transmission of acute itch. However, the pathophysiology of spinal mechanisms underlying intractable itch such as psoriasis remains unclear. In this study, we aimed to determine whether itch‐responsive GRPR+ neurons contribute to the spinal transmission of imiquimod (IMQ)‐induced psoriatic itch. Methods To generate a psoriasis model, C57BL/6J mice received a daily topical application of 5% IMQ cream on their shaved back skin for 7‐10 consecutive days. GRP+ neurons were inhibited using Cre‐dependent expression of Gi‐designer receptors exclusively activated by designer drugs (DREADDs), while GRPR+ neurons were ablated by intrathecal administration of bombesin‐saporin. Results Repeated topical application of IMQ elicited psoriasis‐like dermatitis and scratching behaviors. The mRNA expression levels of GRP and GRPR were upregulated in the cervical spinal dorsal horn (SDH) on days 7 and 10 after IMQ application. Either chemogenetic silencing of GRP+ neurons by Gi‐DREADD or ablation of GRPR+ neurons significantly attenuated IMQ‐induced scratching behaviors. Conclusion The GRP‐GRPR system might be enhanced in the SDH, and itch‐responsive GRPR+ neurons largely contribute to intractable itch in a mouse model of psoriasis.


| INTRODUC TI ON
Itch (pruritus) is an unpleasant sensation that elicits a desire or reflex to scratch. Accumulating evidence suggests that gastrin-releasing peptide receptor (GRPR)-expressing neurons play a central role in the spinal transmission of chemically induced acute itch. 1,2 In fact, intrathecal (i.t.) delivery of GRP evokes robust scratching behaviors in not only rodents but also primates. [3][4][5] Moreover, we have demonstrated that GRP and glutamate cooperatively activate GRPR + neurons through GRPR and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), regulating acute itch originated by sensory C-fiber activation. 6 Despite scientific knowledge for mechanisms of acute itch, the pathophysiology of intractable itch remains unclear. Given that intractable itch associated with various skin diseases causes serious economic loss globally because standard antipruritic agents have limited effectiveness, there is a strong need to develop novel mechanism-based therapeutics. 1,7 Psoriasis is a chronic inflammatory skin disease that causes rapid turnover of skin cells and produces thick, red, and itchy skin covered with silvery scales. 8,9 Much attention has been focused on inflammatory mechanisms in the skin, while knowledge of spinal mechanisms underlying psoriatic itch remains limited.
In this study, we aimed to determine whether itch-responsive GRPR + neurons contribute to the spinal transmission of imiquimod-induced psoriatic itch.

| Mice
All animal experiments were approved by the Animal Research Mice were housed in plastic cages in a temperature-controlled room (23°C-24°C, 60%-70% humidity) with a 12-h dark/light cycle and provided with water and food ad libitum.

| Psoriatic itch
After shaving fur on the rostral back with an electric clipper, mice received a daily topical application of 62.5 mg Beselna cream (5% imiquimod, IMQ; Mochida Pharmaceutical Co., Ltd.) on their shaved back skin for 7-10 consecutive days. 11,12 The psoriasis area and severity index (PASI) for erythema, scaling, and thickness was scored independently from 0 to 4 (0, none; 1, mild; 2, moderate; 3 severe; and 4, very severe), and the total score was presented. For the evaluation of itch-related scratching behaviors, mice were placed in plastic cages (20 × 12 × 12 cm 3 ) with a small amount of bedding. The number of scratching bouts was measured in 10-min intervals for 40 minutes as reported previously. 5 One scratching bout was defined as lifting the hind paw to scratch the nape regions and then returning the paw to the floor or to the mouth for licking. Analyses were carried out in a blinded fashion.

| RT-qPCR
Mice were euthanized by decapitation, and fresh cervical (C3-5) spinal dorsal horn (SDH) tissue was collected in RNAlater solution (Thermo Fisher Scientific). The TRIzol ® Plus RNA Purification Kit (Thermo Fisher Scientific) was used for the isolation of total RNA from the tissues following the manufacturer's instructions. Briefly, tissues were placed in a 1.5-mL RNase-free tube and homogenized with TRIzol reagent. Chloroform was added to each sample, and samples were then centrifuged at 4°C for 15 minutes. The aqueous phase containing RNA was transferred to a fresh tube, and RNA was isolated using a purification column. Total RNA extract was used for the synthesis of The fluorescence intensities were recorded, and data were normalized to ACTB.

| Drug administration
Bombesin-saporin (Bom-Sap; Advanced Targeting Systems) and blank-Sap (Advanced Targeting Systems) were dissolved in sterile phosphate-buffered saline (PBS) and were administered intrathecally (i.t.) in a volume of 5 μL as described previously. 6 Under isoflurane anesthesia, mice were secured by a firm grip on the pelvic girdle and drugs were injected by lumbar puncture between L5 and L6 vertebrae using a 30-gauge needle fitted with a Hamilton microsyringe.
Clozapine-N-oxide (CNO; Enzo Life Sciences) was dissolved in physiological saline and was administered intraperitoneally (i.p.; 0.3 mg/ kg) to awake mice in a volume of 0.1 mL/10 g body weight or i.t.
(3 nmol) to anesthetized mice in a volume of 5 μL.

| Data analysis
Data are presented as mean ± standard error of the mean (SEM).
Statistical analyses were performed using Student's t test, one-way analysis of variance followed by Tukey's multiple comparison test, or two-way analysis of variance followed by Bonferroni's multiple comparison test, as appropriate. Statistical significance was established at P < .05.