Itch, also known as pruritus, is defined as an unpleasant sensation that triggers the desire to scratch.1,12 Although itch sensation that is experienced acutely can be protective against potential damages and threats in the environment (eg, mosquito bite), chronic itch conditions are pathologic and constitute a burdensome clinical problem.29 Specially, the psychological burden produced by chronic itch is delineated by reports of high incidences of suicidal motivation (21.1%) and psychiatric illnesses (70%).9,25 Although this comorbidity is well recognized, whether it exists in rodents remains largely unexplored. Investigating this comorbidity in rodent models will afford the opportunity to clarify the precise mechanisms underlying behavioral pathologies and to test potential therapeutic agents. Furthermore, most clinical studies have revealed the relevance of mood disorders and chronic itch predominantly in dermatological patients;8,17,18 however, it remains unknown whether skin injury–induced pathological alterations per se also contribute to the development of mood disorders. Thus, the first aim of this research is to characterize the affective consequences of chronic itch over time using a myriad of behavioral tests in a mouse model of chronic itch that mimics “dry skin,” and analyze the relevance of mood impairment and exaggerated itch behavior.
Altered hypothalamic-pituitary-adrenal (HPA) axis function and neuroendocrine responsivity to stress have been extensively observed in mood disorders such as anxiety and depression.3,19,28 Although the exact physiological consequences of aberrant HPA axis functions are not fully defined, it is noteworthy that a successful treatment of mood disorders goes along with a correction of aberrant stress reactivity and HPA dysfunction.4,11 Moreover, remitted patients without fully attenuated HPA axis overdrive had a higher risk of relapse.23,26 These observations have led to the assumption that a primary component in the pathogenesis of mood disorders should critically involve factors that influence HPA activities and functions. Consistently, it has been reported that the reactivity of HPA axis in response to stress is impaired in patients with chronic itch.5,6 Given that chronic itch situations could be envisaged as a potential stress for the organism, the second aim of this study is to test whether mice with chronic itch may exhibit altered HPA axis activities and functions at the levels of endocrine hormones, neuroendocrine–immune interaction, and stress-related mRNA transcripts. We also tested the effect of CRF1 receptor (CRFR1) antagonist antalarmin on mood disturbances and HPA axis dysfunctions in mice with chronic itch because CRFR1 was demonstrated to be critically involved in neuroendocrine and behavioral stress reactivity in preclinical studies.16
In this study, we used AEW (acetone and diethylether followed by water) method to induce “dry skin,” which in turn evoked sustained itch-associated behavior in mice. Using this model, we characterized, for the first time in rodents, the affective consequences of chronic itch and explored the underlying mechanisms. Therefore, our work may open an avenue for the investigation of itch-associated psychiatric disorders and their mechanisms in preclinical studies, and thereby advancing the field of itch research.
Male C57BL/6J mice (6-7 weeks old on arrival and obtained from the Laboratory Animal Center of Chinese Academy of Sciences) were used for this study. They were group-housed (4-5 per cage) with food and water available ad libitum and kept in 12-hour light cycles (on at 07:00 AM). The temperature and relative humidity were maintained at 22 ± 0.5°C and 60% ± 2%, respectively. Animals were habituated to the animal facilities for at least 5 days before any testing and behavioral tests were performed in blind with respect to operation or treatment. All experiments and animal handing were in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC) and approved by the Ningbo University Committee on Animal Care and Use. All efforts were made to minimize the number of animals used and their suffering.
2.2. Induction of chronic dry skin itch and scratching behavior in mice
To induce a mouse model of chronic dry skin itch, we followed a previously established procedure with minor modifications.20 Briefly, animals were shaved over the rostral part of the back 1 day before the start of the experiment. A mixture of acetone and diethylether (1:1) was soaked with cotton and applied to the shaved area for 15 seconds, followed immediately by distilled water for 45 seconds, twice per day (10:00 AM for the first time and 8:00 PM for the second time) for 4 weeks. The control mice were treated with distilled water for 1 minute. The AE mice were treated with acetone and diethylether for 15 seconds, without distilled water. The naive mice were treated with neither acetone/diethylether nor distilled water. To evaluate the skin barrier function in mice, the skin hydration and transepidermal water loss (TEWL) were measured on hairless rostral back of the mouse, using an MPA device (Courage and Khazaka, Cologne, Germany) equipped with Corneometer CM825 (the probe to measure skin hydration) and Tewameter TM300 (the probe to measure TEWL).
Spontaneous scratching behavior was evaluated as described previously.20,22 Briefly, 2 hours before the morning AEW treatment, the mice were placed in the recording arena and were acclimated to the experimental environment for 1 hour, and then, scratching behavior was video-recorded for 1 hour (from 9:00 AM to 10:00 AM, ie, just before the daily morning AEW treatment). One bout of scratching was defined as an episode in which a mouse lifted its paw and scratched the treated area for any length of time until the paw returned to the floor. Bouts of scratching were counted offline by a blinded observer.
2.3. Anxiety-like behavior
Anxiety-like behaviors in mice were evaluated using the light–dark test (LDT), elevated plus maze (EPM), and stress-induced hyperthermia (SIH) as established previously7,13 and described in short as follows.
2.3.1. Light–dark test
The apparatus consisted of light and dark boxes (25 × 18 × 20 cm each) connected by a dark tunnel (9 × 7 × 6 cm). The lit compartment was brightly illuminated (1500 lux). Mice were placed in the dark compartment in the beginning of the test and the time spent in the lit compartment was recorded during 5 minutes.
2.3.2. Elevated plus maze
The clear Plexiglass apparatus consisted of 2 open arms (30 × 6 cm) and 2 enclosed arms (30 × 6 × 15 cm), extending from a center platform and placed 50 cm above the floor. Each mouse, with face toward the closed arm, was placed at the center platform of EPM, and its explorative behavior was recorded for 5 minutes. The results were expressed as total time spent in the open arms.
2.3.3. Stress-induced hyperthermia test
Mice were singly housed on wood shavings in smaller makrolon cages (26 × 21 × 14 cm) 24 hours before testing. The first of 2 rectal temperature measurements served as the basal value (T1) and provided the initial stressor. Fifteen minutes later, the rectal temperature of each mouse was measured a second time (T2). The profile of SIH was defined as the difference between the second measurement and the first one (T2-T1).
2.4. Depression-like behaviors
Depression-like behaviors in mice were assayed by forced swim test (FST), sucrose preference test (SPT), and novelty-suppressed feeding (NSF) as established previously7,14 and described briefly as follows.
2.4.1. Forced swim test
Mice were individually plunged into vertical Plexiglas cylinders (height: 25 cm and diameter: 10 cm), which were filled with water (depth: 10 cm and temperature: 24 ± 1°C), for 6 minutes. Mice were deemed immobile when they floated passively, without motion except for making only necessary movements to keep their heads above the water. The immobility time was recorded during the last 4 minutes of the total 6 minutes trial.
2.4.2. Sucrose preference test
A water bottle containing fresh 1% sucrose solution was placed in the shoebox cage next to an identical bottle that contained tap water. All mice were trained to be habituated to the sucrose preference procedure before the treatment of AEW or AE, thereby establishing a stable baseline of sucrose consumption. The training and testing began just before lights off and continued for 24 hours. To prevent potential location preference for drinking, the position of the bottles was changed every 8 hours. The water and sucrose bottles were weighed before and after the preference test, and bottle weight change was used to determine the amount of water and sucrose fluids consumed. The preference for the sucrose solution was calculated as the percentage of sucrose solution ingested relative to the total amount of liquid consumed.
2.4.3. Novelty-suppressed feeding test
The testing apparatus consisted of a 40 × 40 × 30 cm plastic box with floor covered with 1-cm sawdust. Fast mice were placed in a corner of a square arena where a single pellet of food was placed on a white paper positioned in the box center. The latency to first contact or chew the pellet was recorded within a 5-minute period. This procedure elicits a conflict between the drive to eat the food and the fear of venturing to the center of the arena.
In these assays of anxiety- and depression-like behaviors, some mice experienced different behavioral challenges at different time points (1, 2, 3, and 4 weeks). Several rules were followed as: (1) No mice were conducted twice in the same paradigm during the entire experimental period. (2) At different time points, a given test was performed on independent sets of animals. (3) At least 1 week separated 2 different paradigms performed on a given animal. (4) The FST was performed as the terminal paradigm (ie, no other test was performed on a given animal after it experienced the swimming challenge).
2.5. Depletion of spinal TRPV1+ fibers and paradigm for chronic unpredictable stress
To deplete spinal TRPV1+ fibers, mice were intrathecally injected with 25 ng resiniferatoxin (RTX; Sigma-Aldrich, St Louis, MO) in volume of 5 µL using a 31-gauge needle connected to a 50-µL Hamilton syringe. Of note, RTX-induced TRPV1 depletion may induce other neuropathic effects such as thermal hypoalgesia and mechanical allodynia, depending on the dose and delivery route of RTX regimen.10,15 Five days later, the mice were subjected to a 4-week AEW treatment or a chronic unpredictable stress (CUS) procedure. The details on CUS procedure are described as follows.
The mice were subjected to the paradigms of stress challenges delineated by Molina et al.21 with minor modifications. Stress was administered to mice once per day over a period of 28 days. The order and detail of stressors are as follows: day 1 (D1): cold swim (10°C, 4 minutes), D2: restraint (1.5 hours), D3: shaking (30 minutes), D4: tail pinch (1 minutes), D5: water deprivation (24 hours), D6: foot shock (30 minutes, 1 mA, 1-second duration, average 1 shock per minute), D7: cold swim (1°C, 6 minutes), D8: restraint (2 hours), D9: food deprivation (24 hours), D10: shaking (45 minutes), D11: tail pinch (2 minutes), D12: social isolation (24 hours, mice were individually housed in another room), D13: water deprivation (24 hours), D14: foot shock (45 minutes, 1 mA, 1-second duration, average 1 shock per minute), D15: cold swim (8°C, 8 minutes), D16: shaking (1 hours), D17: tail pinch (3 minutes), D18: restraint (2.5 hours), D19: food deprivation (24 hours), D20: foot shock (45 minutes, 1 mA, 1-second duration, average 1 shock per minute), D21: restraint (3 hours), D22: water deprivation (24 hours), D23: tail pinch (4 minutes), D24: cold swim (4°C, 10 minutes), D25: social isolation (24 hours, mice were individually housed in another room), D26: shaking (1.5 hours), D27: foot shock (60 minutes, 1 mA, 1-second duration, average 1 shock per minute), and D28: tail pinch (5 minutes).
2.6. Profiles of HPA axis activity
Blood collection was performed from separate sets of mice independent from the behavioral ones. To assess the circadian HPA axis activities of control and AEW mice, 2 batches of mice were killed to harvest blood samples, one at 8:00 AM and the other at 6:00 PM. As an indirect profile of HPA axis activity, adrenal and thymus glands were removed and their wet weights were measured immediately after blood sample collection.
2.7. Profiles of HPA axis functionality
A thorough evaluation of chronic itch as well as drug intervention on various aspects of HPA axis functionality was performed. For acute stress challenge, tail blood samples were collected to assay baseline ACTH and CORT levels at 9:30 AM. Mice were then placed in Plexiglass restrainers (2.5 cm diameter and 9.5 cm long) for 30 minutes, followed immediately or 90 minutes later by a second blood sample collection. For repetitive stress challenge, the 30-minute restraint was performed per day at the same time (9:30 AM) for 5 consecutive days. The blood samples were collected before and immediately after the last restraint challenge.
To assess the HPA axis negative feedback, the glucocorticoid receptor (GR) agonist dexamethasone (DEX; Sigma-Aldrich) was used to perform the DEX suppression test. Control and AEW mice were intraperitoneally injected with either DEX (0.1 mg/kg) or vehicle (saline, 10 mL/kg). Thirty minutes later, all mice were subjected to an acute restraint stress for 20 minutes to challenge the HPA axis. Then, after 70 minutes (ie, 120 minutes after DEX injection), trunk blood was collected for assessment of plasma CORT level. CORT suppression is expressed as (CORT level from vehicle injection − CORT level from DEX injection)/CORT level from saline injection.
To specifically assess the functionality of the pituitary and adrenal glands, systemic CRF and ACTH challenges were conducted in control and AEW mice. Control and AEW mice were subcutaneously injected with CRF (20 µg/kg with a volume of 10 mL/kg) or intraperitoneally injected with ACTH (1 mg/kg with a volume of 10 mL/kg) at 9:30 AM. Thirty minutes later, these mice were decapitated, and blood samples were collected for plasma CORT assays. The CORT increase is expressed as (CORT level from CRF or ACTH injection − CORT level from vehicle injection)/CORT level from vehicle injection × 100%.
To determine whether the altered HPA axis function influences the neuroendocrine–immune interaction, we assayed splenocyte proliferation in response to varying concentrations of lipopolysaccharide (LPS) for cells collected from AEW and control mice immediately after exposure to a stress of 30-minute restraint or in nonstressed control. Spleens of mice were dissected under aseptic conditions and placed in supplemented RPMI medium 1640 (GIBCO) containing 10 mM Hepes, 2 mM glutamine, and 50 µg of gentamicin per mL. Red blood cells were eliminated by addition of 2 mL of lysis buffer (0.28 M NH4Cl, 0.02 M KHCO3, and 0.01 M EDTA) for 1 minute. After 3 washes, viable mononuclear cells were counted using a hemocytometer and diluted to a concentration of 5 × 106 cells per ml in a medium containing 5% heat-inactivated fetal calf serum. Cells were plated in triplicate in 96-well plates (5 × 105 cells per well) supplemented with LPS from E. coli (O26:B6) across 4 final concentrations of 0, 4, 8, and 16 µg/mL. The cells were left to incubate for 48 hours at 37°C under a 5% CO2/95% air atmosphere. At 44 hours of incubation, plates were pulsed with 1 µCi of [3H]thymidine in 50 µL of medium per well and allowed to incubate for 4 more hours. Cells were harvested onto glass filters using a multiple cell harvester, and radioactivity was quantified in a liquid scintillation counter (PerkinElmer, Boston, MA). The results were expressed as means of cpm for [3H]thymidine incorporation.
2.8. Brain punching and q-PCR
For gene expression analysis, mice were killed by decapitation and brains were quickly removed and frozen at −80°C. Brain sections (300 µm), at levels containing the regions assayed according to the mouse brain atlas delineated by Paxinos and Franklin,24 were made in a cryostat (−20°C). Punches containing the hypothalamic paraventricular nucleus (PVN, −0.6 mm from Bregma), pituitary (PT, −3.3 mm from Bregma), frontal cortex (FC, 1.9 mm from Bregma), hippocampus (HIPP, −2.4 mm from Bregma), central nucleus of the amygdala (CeA, −0.8 mm from Bregma), basolateral amygdala (BLA, −0.8 mm from Bregma), and nucleus accumbens (NAc, 1.8 mm from Bregma) were collected from the brain sections (2-3 punches per section and were merged into one sample) using a microdissecting needle of a suitable size and stored at −80°C.
Total RNA (0.2-0.7 µg per sample) was isolated from brain tissue micropunches using Tri Reagent solution (Thermo Fisher Scientific, Shanghai, China) and subsequently retrotranscribed to cDNA. Quantitative analysis of the relative abundance of CRF, AVP, GR, MR, CRFR1, and AVPR1b gene expression was performed using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA). Samples were run in triplicate for these target genes and for GAPDH as the endogenous control. The data for each target gene were normalized to the reference gene DAPDH, and the fold change in target gene mRNA abundance was determined using the 2(−ΔΔCt) method.
2.9. Pharmacological treatments
The treatment with antalarmin (Tocris Bioscience, Bristol, United Kingdom) began 4 weeks after the AEW treatment onset. For chronic treatment, mice received daily oral antalarmin (5 and 15 mg/kg) or vehicle (10 mL/kg) for 3 weeks (ie, from day 28 to day 48, after the onset of AEW treatment). One day after the last antalarmin treatment (ie, day 49), behavioral tests and HPA axis function assay were performed. To study the consequence of acute antalarmin treatment, behavior tests were conducted 1 hour after antalarmin administration on day 28.
2.10. Statistical analysis
All values are presented as the mean ± SEM. A 2-way repeated-measures analysis of variance (ANOVA) was used and followed by the Tukey test to detect interactions between test groups and time points or interactions between test groups and drug intervention. When needed, multifactor ANOVA was used, for example, in the assay of splenocyte proliferation (AEW and stress were performed as the between-group factors, with the LPS concentration as a within-subject factor). The correlative relationship between itch-related (scratching) behavior and anxiogenic/depressive behaviors was evaluated using Pearson correlation coefficient analysis. Statistical significance was set at P < 0.05.
3.1. Repetitive AEW but not AE treatment caused persistent scratching behaviors in mice
AEW treatment induced marked scratching behavior that lasted at least 7 weeks (P < 0.01, n = 10-12; Fig. 1A), but it was not the case for AE and control treatments. Of note, both AE and AEW treatments evoked disruption of cutaneous barrier function with similar degree, assessed by skin hydration (P < 0.01, n = 10-12; Fig. 1B) and transepidermal water loss (TEWL, P < 0.01, n = 10-12; Fig. 1C).
3.2. Anxiety-like phenotypes in AEW mice
No difference in anxiety-related parameters between naive, control, and AE mice was present throughout the entire experimental period. Regarding AEW mice, they showed time-dependent anxiety-like phenotype in the behavioral tests. In the LDT, AEW mice showed reduced time in the lit part, at 2, 3, and 4 weeks after AEW treatment onset (P < 0.05 for 2 weeks and P < 0.01 for 3 weeks and 4 weeks, n = 10-12; Fig. 2A). In the SIH test and EPM test, AEW mice exhibited anxiety-like behaviors, shown as increase in [INCREMENT]T (P < 0.05 for 3 weeks and P < 0.01 for 4 weeks, n = 10-12; Fig. 2B) and reduced time in the open arm (P < 0.01, n = 10-12; Fig. 2C) respectively, at 3 and 4 weeks after AEW treatment onset but not at earlier time points.
In the correlation analysis, the itch-like behavior and anxiety-like behaviors were analyzed in these same mice at the time point of 4 weeks after AEW treatment onset. The results show that more counts of scratches correlated with a less time spent in the lit part assayed in the LDT (P < 0.01, n = 11; Fig. 2D, left panel), a more [INCREMENT]T assayed in the SIH (P < 0.01, n = 11-12; Fig. 2D, middle panel), and a less time spent in the open arm assayed in the EPM (P < 0.01, n = 11-12; Fig. 2D, right panel), respectively, demonstrating a comorbid relevance between itch-like behavior and anxiety-like behavior in these mice.
3.3. Depressive-like phenotypes in AEW mice
There was no difference in depression-related parameters between naive, control, and AE mice throughout the entire experimental period. For AEW mice, the condition of persistent itch induced time-dependent depression-like phenotype in the behavioral tests. In the FST and SPT, AEW mice displayed depression-like behaviors, shown as increased immobility time (P < 0.01, n = 10-12; Fig. 3A) and reduced percentage of sucrose preference (P < 0.01, n = 10-12; Fig. 3B), respectively, 4 weeks after AEW treatment onset but not at earlier time points. In the NSF, AEW mice showed increased latency to feed 3 weeks after AEW treatment onset (P < 0.05 for 3 weeks and P < 0.01 for 4 weeks, n = 10-12; Fig. 3C).
In the correlation analysis, more counts of scratches correlated with a more time of immobility in the FST (P < 0.01, n = 11; Fig. 3D, left panel), a less percentage of sucrose preference in the SPT (P < 0.01, n = 10; Fig. 3D, middle panel), and a delayed latency to feed in the NSF (P < 0.01, n = 11; Fig. 3D, right panel), respectively, demonstrating a comorbid relevance between itch-like behavior and depression-like behavior in these mice.
Table 1 summarized the tests and results pertinent to the affective condition of control and AEW mice. It was shown that AEW mice developed behavioral phenotypes of anxiety and depression in a time-dependent manner. Moreover, we evaluated whether chronic itch may affect motor function and locomotor activity. Compared with control mice, AEW mice did not show any altered profiles in the rota-rod test and locomotor activity test (Supplementary Fig. 1, available at http://links.lww.com/PAIN/A606).
3.4. Selective ablation of TRPV1 expression in the mouse spinal cord reduced AEW-evoked itch and mood impairment, without influencing mood deficits induced by chronic unpredictable stress
To further test the assertion that the mood deficits of AEW mice are dependent on the condition of persistent itch, we blocked peripheral itch signal by depleting spinal TRPV1-expressing fibers with RTX. Congruent with previous reports,22,31 the TRPV1 level in mouse spinal cord was dramatically reduced by pretreatment of RTX (P < 0.01, n = 6-8; Fig. 4A). Five days after injection of RTX, the mice were challenged with 4 weeks of AEW or CUS procedures (as shown in Fig. 4B). As expected, the scratching behavior evoked by AEW treatment was remarkably reduced by spinal TRPV1 depletion (P < 0.01, n = 10-12; Fig. 4C, left panel). Strikingly, we found that the mood impairment induced by chronic itch was greatly attenuated by spinal TRPV1 depletion, evidenced by increased time in open arm in EPM (P < 0.01, n = 10-12; Fig. 4C, middle panel) and reduced immobility time in FST (P < 0.01, n = 10-12; Fig. 4C, right panel). However, RTX pretreatment may directly impinge on CNS to exert antidepressant or analytic actions. To exclude this possibility, we assessed whether spinal TRPV1 depletion affects CUS-induced anxiety- and depression-like behaviors. Our results show that the 4-week procedure of CUS challenge in mice led to obvious anxiety- and depression-like phenotypes. These symptoms of mood disruption were not modified by the pretreatment of spinal RTX injection (Fig. 4D). Thus, these results support the assumption that the mood impairment of AEW mice should result from the persistent condition of excessive itch sensation.
3.5. Chronic itch impaired HPA axis function but did not alter circadian HPA activity
Because HPA axis is casually involved in the pathogenesis of mood disorders, we determined whether the condition of persistent itch may impact on the HPA activity and functionality. We chose the time point of 4 weeks after treatment onset for analysis because at this time, AEW mice showed the comorbidity of itch and mood impairment. At 4 weeks after treatment onset, AEW mice showed normal basal plasma ACTH (Fig. 5A) and CORT (Fig. 5B) levels at circadian nadir and peak, suggestive of an intact HPA axis activity at physiological condition. Consistent with these results, we did not observe any difference in the adrenal and thymus weights, as well as body weight changes, between control and AEW mice (Supplementary Table 1, available at http://links.lww.com/PAIN/A606), indicating a normal index of previous cumulative levels of ACTH and CORT in AEW mice.
When challenged to acute restraint stress, AEW mice showed delayed recovery of HPA endocrine responses, as reflected by prolonged ACTH (P < 0.01, n = 10-11; Fig. 5C, left panel) and CORT (P < 0.01, n = 10-11; Fig. 5C, right panel) surge, although the peak levels of ACTH and CORT in AEW mice seem not to differ from that in control mice. The habituation to repetitive homotypic stress is a physiological adaptation of HPA axis to stress. Compared with control mice, AEW mice showed blunted HPA axis habituation to repetitive restraint stress, shown as excessive ACTH and CORT levels immediately after the last restraint stress (P < 0.01, n = 10-11; Fig. 5D).
In the dexamethasone (DEX) suppression test, AEW mice showed blunted CORT suppression response to DEX challenge (P < 0.01, n = 10; Fig. 5E), suggestive of a disrupted function of HPA axis feedback regulation. In the CRF challenge test, AEW mice showed blunted CORT surge response to CRF challenge (P < 0.01, n = 11; Fig. 5F), indicating the altered CRF signaling in the HPA axis. In the ACTH challenge test, AEW mice showed normal CORT response to ACTH (Fig. 5G), implying intact endocrine reactivity at the level of adrenal gland.
Because HPA axis has bidirectional connection with the immune system, we asked whether the altered HPA axis functionality, that shown in AEW mice, could be extended to the domain of neuroendocrine–immune interactions. We thus assayed splenocyte proliferation in response to varying concentrations of LPS (E coli O26:B6) for cells collected from AEW and control mice immediately after exposure to a stress of 30-minute restraint or in nonstressed (control) condition. In nonstressed condition, control and AEW mice showed similar degree of splenocyte proliferation in response to varying concentrations of LPS (4, 8, and 16 µg/mL, Fig. 5H). In stressed condition, however, AEW mice showed increased sensitivity of lymphocytes to stress-induced suppression of proliferation, evidenced by suppression of splenocyte proliferation at lower LPS concentrations (4 and 8 µg/mL) in AEW mice compared with that (16 µg/mL) in control mice (P < 0.01, n = 10-12; Fig. 5H). These results reveal the disruption of HPA axis function at the level of neuroendocrine–immune interaction in AEW mice.
3.6. Chronic itch altered gene expression associated with HPA axis regulation
The mRNA expression of several stress-associated mediators was measured in brain regions that have been implicated in HPA functioning and mood regulation. These brain regions assayed are hypothalamic PVN, pituitary, FC, hippocampus (HIPP), central nucleus of the amygdala (CeA), basolateral amygdala (BLA), and nucleus accumbens (NAc). In the PVN, the CRF mRNA level of AEW mice increased remarkably (P < 0.01, n = 10; Fig. 6A), whereas the GR mRNA level decreased compared with that of control mice at the time points of circadian nadir and peak (P < 0.01 for nadir and P < 0.05 for peak, n = 10; Fig. 6A). In the pituitary, AEW mice showed reduced CRFR1 mRNA level at the time points of circadian nadir and peak (P < 0.01, n = 11; Fig. 6B), and reduced GR expression at the time point of circadian nadir but not peak (P < 0.05, n = 11; Fig. 6B). In all corticolimbic regions assayed (FC, HIPP, CeA, BLA, and NAc), the CRF mRNA level of AEW mice was elevated compared with that of control mice at the time points of circadian nadir and peak (P < 0.01, n = 9-12; Figs. 6C–G), strongly implying a prominent overdrive of central CRF system for the AEW mice.
3.7. Repetitive cotreatment with antalarmin, a selective CRFR1 antagonist, ameliorated AEW-induced mood impairment and HPA dysregulation, without influencing the aberrant itch sensation
Finally, we measured the effect of antalarmin, a selective CRFR1 antagonist, on the behavioral symptoms of anxiety and depression in AEW mice, as well as their itch-like behavior. For acute treatment, antalarmin did not show any beneficial effect on the mood impairment and itch-like behavior in AEW mice (Supplementary Fig. 2, available at http://links.lww.com/PAIN/A606). For chronic treatment, antalarmin was administered to mice once per day for 3 weeks (from day 28 to day 48 after the onset of AEW treatment, Fig. 7A). After 3 weeks of treatment, antalarmin did not alter the itch-related behavior in both control and AEW mice (Fig. 7B). However, the same antalarmin regimen attenuated in AEW mice pronounced anxiety- and depression-like behaviors (P < 0.01, n = 10-12; Fig. 7C for anxiety-like behaviors; P < 0.01, n = 10-12; Fig. 7D for depression-like behaviors) in a dose-related manner.
We also evaluated the effect of chronic antalarmin treatment on the impaired HPA functionality in AEW mice. In the DEX suppression test and CRF challenge test, chronic treatment of AEW mice with antalarmin corrected their blunted CORT response to both DEX (P < 0.01, n = 10-11; Fig. 7E, left panel) and CRF (P < 0.01, n = 10-11; Fig. 7E, right panel), and these effects are also dose-dependent. In the acute restraint stress challenge, repetitive antalarmin treatment corrected the delayed CORT surge of AEW mice to normal level (P < 0.01, n = 10-12; Fig. 7F, left panel). In the repetitive restraint stress challenge, chronic treatment of AEW mice with antalarmin normalized their disrupted adaptation of CORT response to repetitive restraint stress (P < 0.01, n = 10-11; Fig. 7F, right panel). Finally, regarding the aberrant neuroendocrine–immune interaction shown in AEW mice, chronic antalarmin treatment corrected their hypersensitivity of splenocyte to stress-induced suppression of proliferation, without altering the measures in nonstress condition (P < 0.01, n = 10-11; Fig. 7G).
This article is the first report demonstrating that chronic itch can dampen mood in rodents. Although the comorbidity of mood disorders and chronic itch is frequently observed in clinical studies,8,17,18 there is no animal study to investigate the affective consequences of chronic itch. This makes for a primary rationale for conducting this study. Furthermore, itch has its own affective component. For example, itch can induce conditioned place aversion in mice.22 It is therefore intriguing to know whether long-term interference of this component has pathological impact on mood. With this regard, we first established mouse chronic itch model using AEW method that models “dry skin,” because it is widely used as a canonical animal model of chronic itch.2,30,33
AEW mice developed anxiety-like symptoms 2 to 3 weeks and depression-like phenotypes 3 to 4 weeks after the AEW treatment onset, indicating that mood impairments due to chronic itch evolve over time. The synchronous symptoms of pruritus and mood disturbance in AEW mice suggest that the mouse AEW model is suitable for mimicking the comorbidity of chronic itch and mood disorders. Because some of the present mood-related behavior tests, such as EPM and FST, are motor- or locomotor-dependent, an important caveat is that the motor function and locomotor activity of AEW mice were not spoiled by chronic itch. This possibility is grossly ruled out by the fact that chronic treatment of mice with AEW did not modify the profiles in rota-rod test and locomotor activity. Furthermore, it is interesting to know whether chronic itch can impact on other neurological aspects relevant to psychiatric disorders, such as memory, sleep, and social interaction, because these comorbidities are also frequently observed in patients with chronic itch.17,25,27,32
Regarding clinical relevance of psychiatric disorders and chronic itch in the context of dermatological disorders such as psoriasis, atopic dermatitis, and eczema,8,17,18 a key issue is raised as to whether skin injury–induced pathological alterations also contribute to the development of mood impairment. Here, we can tentatively address the issue from several aspects. First, all measurements of AEW mice in anxiety- and depression-like behaviors are statistically related to their itch-associated behavior in the correlation analysis. Thus, the correlative relationship between itch-related behavior and anxiogenic/depressive behaviors was firmly established. Second, the acetone and diethylether (AE)-treated mice (devoid of exaggerated scratching behavior) did not develop symptoms of mood disturbance, notwithstanding similar degree of skin barrier deficits for AEW and AE mice. Indeed, our results that AE mice did not develop itch-associated behavior are congruent with the report by Miyamoto et al.,20 who firstly established the AEW model of chronic itch. Third, the symptoms of mood impairment in AEW mice were exclusively attenuated by chemical depletion of spinal TRPV1-expressing fibers, which can significantly reduce their exaggerated itch sensation. This outcome suggests that the mood deficits of AEW mice are dependent on the condition and degree of persistent itch. Together, these results support a direct connection between chronic itch and mood disorders. This assertion is consistent with clinical observations25,32 and highlights the importance of mitigating the provocative itch hypersensitivity in dermatological patients with chronic itch.
The HPA axis is a predominant system responsible for maintaining homeostasis in response to stress and plays a causal role in the pathogenesis of stress-related disorders such as depression and anxiety.3,4,11,19,28 Overall, our results argue for the hypothesis of HPA axis dysfunction in the condition of chronic itch, which incorporates several characteristic aspects. (1) AEW mice showed normal ACTH and CORT responses immediately after an acute heterotypic (restraint) stress, but a depressed recovery 90 minutes after the stress. (2) Chronic itch impaired the habituation of neuroendocrine response to repetitive homotypic stress, evidenced by the attenuated hormone drop in response to repetitive homotypic stress in AEW mice. (3) AEW mice showed blunted CORT responses to both dexamethasone and CRF but not to ACTH, indicating their impaired HPA feedback regulation and CRF signaling with a normal neuroendocrine reactivity at the level of adrenal gland. (4) AEW mice showed increased sensitivity of lymphocytes to stress-induced suppression of proliferation, suggestive of their dysfunction of HPA axis at the level of neuroendocrine–immune interaction. Considering the altered immune reactivity of AEW mice to a low dose of endotoxin (LPS), it is interesting to ask whether chronic itch affects long-term predisposition to inflammation. Collectively, these observations point to a primary disturbance of HPA axis function in AEW mice with chronic itch. It is thus feasible to suppose that dry skin–induced pathological alterations in itch processing within peripheral nervous system and central nervous system, which manifest as exaggerated scratching behavior, should be translated into surrogate markers of sustained stress with subsequent impairment of HPA axis function, and thereby leading to behavioral phenotypes of mood disturbance.
We further asked whether parameters of HPA functionality at the level of mRNA transcripts are altered in stress-related brain regions of AEW mice. Our results demonstrate a predominant CRF mRNA upregulation of AEW mice in the target brain regions. This outcome indicates an aberrant CRF signaling in the brain of AEW mice, which in turn may dampen the circuit controlling neuroendocrine and behavioral effectors that impact on the phenotypes characteristic of mood disorders. Another remarkable alteration is the decreased gene expression of GR in the PVN and pituitary. This outcome may account for the intensified neuroendocrine response to both acute and repetitive restraint stress because the main function of GR in the brain during stress seems to suppress stress-induced overdrive of the HPA axis at the level of the PVN and anterior pituitary.3,28 Also, it may explain the flattened HPA axis feedback efficacy seen in AEW mice, reflected by blunted CORT suppression response to DEX challenge. Finally, we observed a CRFR1 gene downregulation in the pituitary of AEW mice, but not in the corticolimbic regions such as hippocampus, CeA, and NAc. Given that AEW mice showed normal circadian levels of ACTH and CORT, the decreased expression of CRFR1 mRNA in the pituitary may represent an adaptation and feedback regulation in response to the elevated CRF level. Combined with the results that AEW mice showed normal CORT response to ACTH, it seems logical to assume that the process of HPA axis adaptation, reflected by normal circadian ACTH and CORT levels, may be achieved eventually at the level of the adrenal gland, despite the fact that adrenal, pituitary, and PVN mechanisms may act synergistically to realize the concept of HPA dysfunction. Together, these results indicate that AEW-induced chronic itch is associated with several, but selective, changes in molecular targets that participate in stress reactivity and in the pathogenesis of mood disorders.
Finally, we assessed the therapeutic potential of antalarmin, a CRFR1 antagonist, on chronic itch-induced mood disorders in mice, given that AEW mice displayed a marked overdrive of CRF tone in the brain. Our data clearly show that antalarmin possessed therapeutic efficacy against mood disorders in the context of chronic itch. After chronic treatment, antalarmin ameliorated the behavioral phenotypes of anxiety and depression in AEW mice, without influencing the profiles in control mice. The selective pharmacological action of antalarmin in AEW mice implies that CRFR1 may share a promising therapeutic target for the development of pharmacological agents to treat itch-associated psychiatric disturbance. Of note, the same antalarmin regimen did not alter the itch-associated behaviors in the AEW mice, indicating that perturbations of the HPA system may not be the primary cause of increased scratching performance in AEW mice. Because the restoration of the HPA axis regulation is closely linked to a successful treatment of mood disorders,4,11 we also evaluated the effect of chronic antalarmin treatment on the putative AEW-induced disruption of HPA functioning. Our findings show that chronic antalarmin treatment can correct abnormal HPA axis functions at the level of endocrine hormones and neuroendocrine–immune interaction. In light of these results, it is plausible to assume that antalarmin, a drug sharing a selective property of CRFR1 antagonizing, can offset the overdrive of CRF signaling in the brain of AEW mice, and thereby enabling the dysfunctional HPA axis to restore the physiological control over stress reactivity system, then allowing for recovery from mood impairment. However, it should be noted that although CRFR1 antagonists possessed promising efficacy against depressive-like behaviors in animal studies, they failed to translate to humans.16
In this study, we present an experimental system for studying itch-induced mood disturbances and find, for the first time, that chronic itch can lead to behavioral phenotypes of anxiety and depression in rodents. We also reveal that impairment of HPA axis function could be a primary etiologic factor or a pathophysiological correlate in the development of mood disorders in the context of chronic itch. These findings provide a mechanistic framework for exploring the affective consequences of chronic itch. Finally, we provide evidence showing the beneficial effect of antalarmin, a selective CRFR1 antagonist, on chronic itch-induced mood disturbances and HPA axis dysfunctions. It is meaningful and implies that drugs acting at CRFR1 may possess therapeutic potential on itch-associated psychiatric disorders. Together, our findings may open a novel avenue for the study of affective consequences of chronic itch, as well as the underlying mechanism and potential interventional strategy.
Conflict of interest statement
The authors have no conflict of interest to declare.
This work was sponsored by National Basic Research Program of China (2015CB553504), National Natural Science Foundation of China (81541087, 31270028 and U1132602), Natural Science Foundation of Zhejiang Province (LY18H310006), Innovative Research Team of Ningbo (2015C110026), Key Research and Development Project of Hunan Science and Technology Department (2017SK2152), Science and Research Project from Health and Family Planning Commission of Hunan Province (B20180265), and K. C. Wong Magna Fund in Ningbo University.
The authors thank Dr Xiang-Yu Cui and Dr Yu Yang for insightful comments on this work and manuscript.
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