Neuropathic pain drives anxiety behavior in mice, results consistent with anxiety levels in diabetic neuropathy patients

Background: Epidemiological studies in patients with neuropathic pain demonstrate a strong association with psychiatric conditions such as anxiety; however, the precipitating pathology between these symptoms remains unclear. To investigate this, we studied the effects of lifelong stress on levels of neuropathic pain–like behavior and conversely, the effects of chronic neuropathic injury on anxiety-like status in male and female mice. In addition, we assayed this link in painful and painless diabetic peripheral neuropathy patients. Methods: Male and female mice were subject to ongoing life-stress or control living conditions. Baseline sensitivity and anxiety tests were measured followed by spared nerve injury (SNI) to the sciatic nerve. Subsequent sensory testing occurred until 3 weeks after SNI followed by anxiety tests between 4 and 6 weeks after SNI. Results: Levels of tactile or cold allodynia did not differ between adult mice subject to lifelong chronic stress, relative to nonstressed controls, for at least 3 weeks after SNI. By contrast, longer-term neuropathic mice of both sexes displayed pronounced anxiety-like behavior, regardless of exposure to stress. If sex differences were present, females usually exhibited more pronounced anxiety-like behavior. These ongoing anxiety behaviors were corroborated with plasma corticosterone levels in distinct animal groups. In addition, data from patients with painful and nonpainful diabetic neuropathy showed a clear relationship between ongoing pain and anxiety, with females generally more affected than males. Discussion: Taken together, these data demonstrate a strong link between chronic neuropathic pain and chronic anxiety, with the driver of this comorbidity being neuropathic pain as opposed to on-going stress.

All animal procedures were approved by the Boston Children’s Hospital Animal Care and Use Committee, under animal protocol numbers 15-04-2928R and 16-01-3080R. All experiments were conducted in a blinded fashion in a quiet room from 09:00 to 18:00. Male and female adult C57BL/6 mice (delivered 6–7 weeks for use at 9 weeks) were obtained from Charles River Labs (CRL). Time-mated female mice were delivered 1 week before birth from CRL. Special care was taken to not disturb the mice (eg, cage changing) on test days, to avoid transient stress confounds. Mice were housed with their littermates (up to 5 mice per cage) in OptiMICE cages with food and water ad libitum (temperature 22 ± 1°C, 50% relative humidity, lights on from 07:00 to 19:00). Each behavioral test was performed on 2 independent cohorts of male and female mice, the data checked for consistency then merged. Figure ​Figure11 shows the timeline for mice that were exposed to ongoing life stress or left alone, before spared nerve injury (SNI)-induced neuropathic pain. Baseline sensitivity tests were measured before SNI injury (in 7–8-week-old mice) and at defined points following it. With the exception of 2 weeks in early life, chronically stressed animals were continuously subject to maternal separation (MS) or chronic mild stress (CMS) throughout the experiment. Plasma corticosterone levels were measured in 3 distinct groups of mice, naive (nonstressed); mice with SNI; or mice that underwent MS followed by CMS. Each was age matched to the SNI results and taken at 4 to 5 weeks after nerve injury. Experimental timeline for male and female mice differentially subjected to ongoing life stress (A) or control unstressed mice (B) before SNI. Baseline sensitivity tests were measured before SNI and anxiety tests were performed before and after SNI within the time frames shown. A separate group of animals were used for plasma blood draws and these assays were measured between 4 and 5 weeks after SNI and within the same time frame for control and ongoing life-stress animals. SNI, spared nerve injury. From P5, pups were separated from their mother’s home cage and placed on a warm surface (30°C) for 2 hours between 11:00 and 13:00 each day and then returned to their home cage dams. This procedure was repeated daily until P19. At P21, the mice were separated by sex and housed with their littermates up to n = 5 per cage. After this, the mice were left for 1 week until P29 (4 weeks). Previously, maternally separated mice were subject to CMS from 4 weeks of age. One of 8 stressors was randomly performed on each cage of animals every day for 6 of the 7 days in the week based on previous studies.27,44 The stressors were: (1) cage shake (horizontal shaking, 5 minutes); (2) cold swim (5 minutes per mouse); (3) cage tilt (45° for 8 hours); (4) space reduction (50% cage space for 8 hours); (5) moist bedding (400 mL of water tipped onto bedding and mice left for 8 hours); (6) overnight food deprivation (16 hours); (7) daytime water deprivation (8 hours); and (8) overnight illumination (lights on from 19:00 to 07:00 in addition to the normal day light period). Once the list of 8 interventions was complete, a new random run of the 8 different interventions was performed and this procedure was repeated until the end of the experiment. Mice were anesthetized with isoflurane (2%–4%) at 9 weeks and SNI surgery performed; the tibial and common peroneal branches of the sciatic nerve were tightly ligated with a silk suture and transected distally, whereas the sural nerve was left intact.4,8 The behavioral time course of tactile and cold sensitivity before and after SNI in C57BL/6 mice was measured as in Refs. 4,8. Mice were tested twice at baseline and then 7, 14, and 21 days after SNI. For the CMS cohort, the mice were not subject to the CMS procedure on the day of testing. Mechanical allodynia was measured using von Frey filaments (Touch-Test Sensory Evaluators; North Coast Medical, Inc, Gilroy, CA) ranging from 0.02, 0.04, 0.07, 0.16, 0.4, 0.6, 1, and 2 g, respectively. Each filament was tested 10 times in increasing order starting with the filament producing the lowest force. Von Frey filaments were applied at least 3 seconds after the mice had returned to their initial resting state. For the baseline mechanical sensitivity test, all filaments were applied and the number of withdrawals was recorded. For tactile allodynia, the minimal force filament for which animals presented either a brisk paw withdrawal and/or an escape attempt in response to at least 5 of the 10 stimulations determined the mechanical response threshold. Cold allodynia was assayed by applying a 5 μL drop of acetone to the hind paw, ipsilateral to SNI injury, and measuring the amount of time the animal spent flinching/licking/biting the paw in seconds. The elevated plus maze consisted of 2 open and 2 closed arms each opposite its counterpart, extended from a central platform to create a plus shape elevated 50cm above the floor. Mice were placed on the center platform of the maze, facing a closed arm, and allowed to explore the apparatus for 5 minutes. The percent of time spent in open arms was used as a surrogate measure of anxiety-like states.30 Distance travelled in the closed arms was considered as a measure of locomotor activity. The apparatus consisted of a square arena surrounded by a wall. Sunken into the arena floor were 9 holes equally spaced in 3 rows. Activity (horizontal movement and investigation of the holes) was detected by infrared beams and recorded by a computer. The test duration was 15 minutes and was initiated by placing a mouse into the periphery of the arena. Total distance traveled in the periphery (ambulation), total time spent in the center (nonanxious behavior), and total number of nose pokes into the holes (exploratory behavior) were assessed. The apparatus consisted of a 2-compartment chamber connected by a small aperture that allowed free access to both sides. The test began by placing a mouse in the dark compartment and recording behavior; time spent in the lit compartment (nonanxious behavior) and distance traveled in the light zone (ambulation) were recorded. Plasma corticosterone levels were measured in 3 distinct groups of mice, naive (nonstressed); mice with SNI; or mice that underwent MS followed by CMS. Each of these groups was further divided into mice that underwent acute restraint stress or remained unstressed in the home cage before blood collection. Restraint stress consisted of mice inserted into 50 mL conical polypropylene tubes for 5 minutes. The tube had holes placed along its length and the conical face removed to aid breathing. After restraint, the mice were returned to their home cage for 20 minutes and then euthanized with carbon dioxide inhalation before cardiac puncture to collect blood samples, stored in 3 mL EDTA tubes. Unrestrained mice were left in their home cage undisturbed before blood collection. All mice were euthanized in a room separated from that of housing before sample collection. All samples were collected between 11:00 and 13:00. Plasma was prepared from the blood samples and stored at −80°C until required. CORT was quantified by ELISA (#ADI-900-097; Enzo Life Sciences, Farmingdale, NY) according to the manufacturer’s instructions. Data are presented on 176 adult patients with diabetes mellitus who participated in the Pain in Neuropathy Study (PiNS), a cross-sectional observational multicenter study in the United Kingdom designed to determine the somatosensory phenotype of painful and painless diabetic peripheral neuropathy. The total cohort consists of 191 participants; however, 15 did not complete the anxiety questionnaire. Information on study methodology and sample has been published.39 Participants underwent a neurological examination, quantitative sensory testing, nerve conduction studies, and skin biopsy for intraepidermal nerve fiber density assessment. Toronto Clinical Scoring System (TCSS) was used as a screening tool for diabetic peripheral neuropathy and correlates with diabetic neuropathy severity.6 Participants also completed a series of questionnaires assessing the presence of pain, pain intensity, pain distribution, and psychological and functional impact of pain. The Depression Anxiety Positive Outlook (DAPOS) measure31 was used to assess anxiety; a 7-day pain diary to assess ongoing neuropathic pain intensity39; TCSS as a measure of diabetic neuropathy severity; HbA1c (glycated haemoglobin-A1c concentration as a measure of average plasma glucose concentration over the preceding 120 days); age; and body mass index (BMI). Sample size was determined according to the warm detection threshold for patients with diabetes (Shun et al., 2004), which revealed that a minimum sample size of 34 was required per group for a power of >0.8, SPSS Statistics Version 22 (IBM). Data were tested for normality with the D’Agostino-Pearson normality test and by visual inspection. Data were not normally distributed and are reported as median with interquartile range. Data were compared across multiple groups with the Kruskal–Wallis test (the Dunn post hoc test). In the case of comparisons between 2 groups only, the nonparametric Wilcoxon rank-sum test was used. Spearman correlation analyses were performed to explore associations between DAPOS anxiety and pain intensity. A multivariate analysis was performed to control for potential confounding factors. Ranked nonparametric multivariate analysis of variance (ANOVA) with the Wilk’s Lambda statistic and multivariate principal component analysis were performed. All values were normalized and scaled before analysis. Statistical significance set at P < 0.05.

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