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. Author manuscript; available in PMC: 2021 Jul 1.
Published in final edited form as: Anesth Analg. 2020 Jul;131(1):298–306. doi: 10.1213/ANE.0000000000004652

Differential regulation of the glucocorticoid receptor in a rat model of inflammatory pain

Shaimaa I A Ibrahim 1,2, Judith A Strong 1, Katherine A Qualls 1,2, Yvonne M Ulrich-Lai 3, Jun-Ming Zhang 1
PMCID: PMC7299821  NIHMSID: NIHMS1588906  PMID: 31990732

Abstract

Background:

Anti-inflammatory corticosteroids are a common treatment for different conditions involving chronic pain and inflammation. Clinically used steroids target the glucocorticoid receptor (GR) for its anti-inflammatory effects. We previously reported that GR in sensory neurons may play central roles in some pain models, and that GR immunoreactivity signal in dorsal root ganglia (DRG) decreased after local inflammation of the DRG (a model of low back pain). In the current study, we aimed to determine if similar changes in GR signal also exist in a skin inflammation model, the Complete Freund’s Adjuvant (CFA) model (a model of peripheral inflammatory pain), in which the terminals of the sensory neurons rather than the somata are inflamed.

Methods:

A low dose of CFA was injected into the hindpaw to establish the peripheral inflammation model in Sprague Dawley rats of both sexes, as confirmed by measurements of behavior and paw swelling. Immunohistochemical and western blotting techniques were used to determine the expression pattern of the GR in the inflamed hindpaw and the DRGs. Plasma corticosterone levels were measured with radioimmunoassay.

Results:

The immunohistochemical staining revealed that GR is widely expressed in the normal DRG and skin tissues. Paw injection with CFA caused upregulation of the GR in the skin tissue on post-injection day one, mostly detected in the dermis area. However, paw inflammation significantly reduced the GR signal in the L5 DRG one day after the injection. The GR downregulation was still evident 14 days after CFA inflammation. On day 1, western blotting confirmed this downreguation, and showed that it could also be observed in the contralateral L5 DRG, as well as in the L2 DRG (a level which doesn’t innervate the paw). Plasma corticosterone levels were elevated in both sexes on day 14 after CFA compared to day 1, suggesting autologous downregulation of the GR by corticosterone may have contributed to the downregulation observed at day 14 but not day 1.

Conclusions:

There are distinctive patterns of GR activation under different pain conditions, depending on the anatomical location. The observed downregulation of the GR in sensory neurons may have a significant impact on the use of steroids as treatment in these conditions and on the regulatory effects of endogenous glucocorticoids.

Introduction

Anti-inflammatory corticosteroids are a common treatment for different conditions involving chronic pain and inflammation. Clinically used steroids target the glucocorticoid receptor (GR) for its anti-inflammatory effects, and hence are used in some pain conditions that involve inflammation. The GR is expressed at relatively high levels in the DRGs1, where it is found in sensory neurons1–5. Hence the sensory neuron may be one important target for glucocorticoid effects on pain.

Glucocorticoid receptor levels are regulated by many factors. They are downregulated by endogenous or exogenous glucocorticoids. There is also a complex relationship between pro-inflammatory cytokines and glucocorticoids: glucocorticoid anti-inflammatory effects are importantly dependent on their ability to down-regulate pro-inflammatory cytokines, but pro-inflammatory cytokines can also downregulate the glucocorticoid receptor via multiple cellular and systemic mechanisms6. The latter processes may contribute to glucocorticoid resistance as well as modify the effects of endogenous glucocorticoids in pain conditions.

Previously, in a rat pain model induced by local inflammation of the lumbar DRG (to mimic some forms of low back pain), we observed decreased GR expression in the inflamed DRG2. We wondered if this was specific to that particular pain model, or if a similar process might occur in other inflammatory pain conditions. In the current study, we aimed to determine if similar changes in GR signal also exist in a model of peripheral inflammatory pain, the Complete Freund’s Adjuvant (CFA) model, in which the terminals of the sensory neurons rather than the somata are inflamed. We also examined the effects of this model on the function of the hypothalamic-pituitary-adrenal (HPA) axis, since adrenal steroids are an important endogenous ligand for the GR.

Materials and Methods

The authors have adhered to the applicable ARRIVE guidelines. Additional details about the experimental methods can be found in Supplemental File 1. All surgical procedures and the experimental protocol were approved by the University of Cincinnati institutional animal care and use committee and adhered to the guidelines of the Guide for the Care and Use of Laboratory Animals.

Animals

Adult Sprague-Dawley rats (8 weeks old) were purchased from Envigo (Indianapolis, IN, USA). Male and female rats were used in equal numbers in the experiments. Rats were housed under a controlled diurnal cycle of 14-h light and 10-h dark. The data presented were obtained from 48 male and 48 female rats. The numbers of rats, treatments, and corresponding figure numbers are presented in Supplemental Table 1 in Supplemental File 1.

Procedure for localized paw inflammation with complete Freund’s adjuvant (CFA)

CFA (concentration of 1 mg/ml of Mycobacterium tuberculosis, heat killed and dried, in 85% paraffin oil and 15% mannide monooleate) was diluted with an equal volume of saline. Under brief isoflurane anesthesia, 50 μl of the mixture was injected subcutaneously into the heel region of one paw using an 8 mm 31 gauge needle. For comparison, a dose of 150 μl CFA at 1 mg/ml was shown to cause only local tissue and joint inflammation, with arthritic changes in only the local joints and seen only at later times (e.g., 30 d)7 than we studied in our experiments, whereas higher doses (e.g., 5 mg/ml) are required to elicit systemic inflammation and arthritic changes in more distant and contralateral joints8.

Immunohistochemistry (IHC)

As described previously9 rats were anesthetized with pentobarbital sodium and perfused through the left ventricle of the heart with 0.1 M phosphate buffer, followed by perfusion with 4% paraformaldehyde. DRGs were removed and post-fixed in 4% paraformaldehyde, then transferred to 4% sucrose overnight. Frozen sections of DRG and skin were cut at 10 μm and 30 μm respectively. Sections for IHC were selected randomly for the analysis. To reduce variability, sections from normal and CFA skin or DRG were mounted side-by-side on each slide and processed together.2 Sacrifice of animals for obtaining IHC (or western blot) samples was always conducted starting around 9:00 A.M (i.e. ~3 to 4 hours after lights on).

Sections were permeabilized, blocked with 10% normal goat serum, and incubated overnight at 4°C with primary antibodies followed by secondary antibodies. The primary antibody used was anti-GR (1: 200 dilution, catalog number sc-1004, Santa Cruz, rabbit polyclonal antibody used to identify the activated GR, also known as M-2010, as previously used for neuronal staining2, 11). The antibody used in this study demonstrated reduced staining after genetic or shRNA-mediated GR knockdown in brain neurons from mice and rats11, 12 , 13; and we have previously validated the antibody using siRNA knockdown in the DRG2 under conditions as used in this study. Negative control experiments included the removal of the primary or the secondary antibody. Images from multiple sections of DRG and skin from each animal, selected at random without regard to the amount of signal observed, were captured with a fluorescent microscope. Overall intensity was measured and normalized by the area measured. Areas dominated by neuronal cell bodies in DRG were analyzed, rather than predominately axonal regions. Data from multiple sections was summarized as animal averages and the statistical analysis was applied to these average values. For immunoreactive intensity quantification, intensity/area in treated groups was calculated and normalized to that seen in normal DRG or skin measured side-by-side in the same experiment.

Western blotting

DRGs were homogenized in lysis buffer. 40 μg of sample protein was loaded per lane and proteins were separated by gel electrophoresis. Proteins were then transferred to polyvinylidene difluoride membranes and incubated overnight with the same GR primary antibody as used for immunohistochemistry (1/1000) and mouse anti-glyceraldehyde 3 phosphate dehydrogenase (GAPDH; 1/2000) used as a loading control. The membranes were then washed and GR was probed with anti-rabbit secondary antibody conjugated with horse radish peroxidase (HRP; 1/2000, Cell Signaling, catalog # 7074). Protein bands were visualized by enhanced chemiluminescence. Bands were quantified using Image J (NIH) and GR expression normalized by GAPDH. Data are presented normalized to the average values in normal tissue measured in the same gel (“Normalized GR”) except where indicated.

Plasma corticosterone (CORT) levels and body and organ weights

Blood samples were collected from the tail vein, from awake freely moving animals. To obtain accurate unstressed baseline levels, animals were housed overnight in a room separate from the normal housing (to which they were acclimated for one additional night prior to the experiment), and which was not entered by animal care technicians in the morning. At the desired time points the following day, each cage (2 animals) was quickly moved to an adjacent room and blood obtained from both animals within 3 minutes, not in the presence of other cages or animals. The same animals were used for postoperative day (POD)1 and POD14 measurements, and were then sacrificed after the 8:00 a.m. blood draw on day 14, and the thymus, heart, and adrenal glands dissected out and weighed to look for indicators of chronic hypothalamus-pituitary-adrenal (HPA) axis activation, as adrenal hypertrophy and thymic involution are considered indirect indices of chronically elevated ACTH and glucocorticoid tone, respectively14–16. Heart tissue was included as a negative control tissue whose weight is not expected to vary with HPA activation. Blood was collected into tubes with EDTA and centrifuged, and the plasma was stored at −80°C. Plasma CORT levels were measured using radioimmunoassay (RIA)17. The minimum level of detection for this assay is 7.7 ng/ml, and the intra-assay and inter-assay coefficients of variance are both 7%, as indicated by the manufacturer.

Statistics and data analysis

No animals were excluded from analysis. Sample sizes were based on our previous experience with the procedures used, as detailed in Supplementary Methods section in Supplementary file 1. In general, males and females were used in equal numbers in each group and their data combined because we didn’t see any obvious sex differences in the data. However, CORT levels and related measurements were not combined due to the well-known effects of sex on these measures; sex differences in some of the parameters (e.g. CORT, body weight) are already well-established in the literature18, 19. For immunohistochemical quantification, values obtained from multiple images from each rat were averaged, and the statistical analysis was conducted on these averages using the number of rats as the N value for each group.

Graphpad Prism (La Jolla, CA), version 6 software and SigmaPlot version 14 (Systat Software) were used for statistical analysis. Behavioral time course data and CORT levels were analyzed using two-way repeated measures ANOVA with Holm-Sidak multiple comparisons posttest to determine on which days experimental groups differed. For these analyses, F values for the comparison between groups are given as F (degrees freedom in numerator, degrees freedom in denominator). Comparisons between groups in other experiments were performed with unpaired Student’s t-test. Two-tailed tests were used throughout. Significance was ascribed for p<0.05. Levels of significance are indicated in the figures by the number of symbols, e.g., *, p = 0.01 to <0.05; **, p = 0.001 to 0.01; ***, p < 0.001. Data are presented as mean ± S.E.M.

Results

GR immunoreactivity is enhanced in the skin after local paw inflammation

We used immunohistochemical quantification to examine the GR immunoreactivity in normal and CFA (POD1) inflamed skin tissues. This analysis showed that the GR immunoreactivity was observed in the normal skin tissue (Figure 1A; compare to negative control Figure 1C). However, one day after the CFA inflammation (Figure 1B), the GR immunoreactivity was increased dramatically in the epidermis and the sub-dermis skin tissue (Figure 1B and summary data, Figure 1D).

Figure 1: GR immunoreactivity is enhanced in the skin after local paw inflammation.

Figure 1:

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Examples of immunostaining of glucocorticoid receptor (GR) in sections from (A) Normal skin tissue, (B) Complete Freund’s adjuvant (CFA) injected skin tissue one day after inflammation (sections taken near the injection site), (C) negative control skin tissue (no primary antibody). Scale bar 50 μm. (D) Quantification of GR immunoreactivity showed GR upregulation in the CFA skin tissue one day after inflammation compared to normal skin measured in side-by-side experiments (value normalized to 1). **p < 0.01, significant difference between the groups (unpaired student t-test, p= 0.002). The results shown combine equal number of both sexes (n= 2 males and 2 females per group, 20–30 sections/rat).

In the present study, we re-confirmed that the CFA injection resulted in behavioral effects (static mechanical, dynamic mechanical, and cold allodynia, as well as spontaneous pain) and paw swelling lasting at least 14 days (longest time point studied in the present study), as shown in Supplemental File 2 which contains Supplemental Figure 1. The effects were similar to those observed in our previous study20.

Local paw inflammation unexpectedly reduced GR expression in the DRGs

We used immunohistochemical quantification to examine the GR immunoreactivity in L5 DRGs of normal and CFA (POD1) inflamed rats. This analysis showed that the GR immunoreactivity in the DRG tissue of normal animal (Figure 2A) was evident in the neuronal and non-neuronal cells, compared to the DRG in the negative control (Figure 2C) and as we previously reported2. However, one day after the CFA inflammation (Figure 2B), the GR immunoreactivity was significantly reduced in the L5 DRG (Figure 2D).

Figure 2: Effect of CFA injection on GR immunoreactivity in the L5 DRGs.

Figure 2:

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Examples of immunostaining of glucocorticoid receptor (GR) in sections from (A) normal L5 dorsal root ganglion (DRG), (B) L5 DRG one day after paw inflammation with complete Freund’s adjuvant (CFA), (C) negative control DRG tissue (no primary antibody). Scale bar 50 μm. (D) Quantification of GR immunoreactivity showed GR downregulation in the L5 DRG one day after CFA inflammation compared to normal DRG measured in side-by-side experiments (value normalized to 1). *p < 0.05, significant difference between the groups (unpaired student t-test, p= 0.017). The results shown combine equal number of both sexes (n= 2 males and 2 females per group, 20–30 sections/rat).

Western blot analyses were used to examine the GR protein levels one day after the CFA inflammation in the L5 DRG ipsilateral to the inflamed paw. The western blot of DRG proteins identified a single GR-specific protein band at 90 kDa. Quantification of the band densities showed that GR expression was significantly reduced 1 day after CFA injection, consistent with the IHC data (Figure 3 A, B; IHC values reduced to 39% of control, Western blot values reduced to 29% of control).

Figure 3. Effect of CFA injection on GR expression in the DRGs on day 1.

Figure 3.

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Detection of glucocorticoid receptor (GR) protein expression in the dorsal root ganglia (DRG) from complete Freund adjuvant (CFA) injected rats one day after inflammation or naïve rats was examined with western blot analysis. A) Examples of observed bands from ipsilateral L5 DRG. CFA and normal samples loaded in alternating lanes as indicated by the labels on the first pair of samples. B) Quantification of GR protein showed reduction of the GR protein in the ipsilateral L5 DRG one day after CFA injection compared to L5 DRGs from normal rats. **, p<0.01, significant difference between the groups (unpaired student t-test, p=0.0025). C) Examples of observed bands from contralateral L5 DRG samples. D) Quantification of GR protein showed reduction of the GR protein in the contralateral L5 DRG one day after CFA injection compared to L5 DRGs from normal rats. **, p<0.01, significant difference between the groups (unpaired student t- test, p=0.0046) E) Examples of observed bands from the ipsilateral L2 DRG (which does not innervate the paw). F) Quantification of GR protein showed reduction of the GR protein in the L2 DRG one day after CFA injection compared to L2 DRGs from normal rats. Protein obtained from the same animals as C and D. **, p<0.01, significant difference between the groups (unpaired student t- test, p = 0.0037). In panels B, D, and F, GR expression levels are normalized to the levels observed in the corresponding DRGs obtained from normal rats and run on the same gel (value set to 1). N = 4 – 6 samples per group. The results shown combine data from both sexes.

The ipsilateral L5 DRG innervates the inflamed region of the paw. We considered that activity in the neurons innervating the paw might convey the signal resulting in downregulation of the GR. However, contrary to this hypothesis, the GR expression was also reduced in the contralateral L5 DRG (Figure 3 C, D), and in the L2 DRG that does not innervate the inflamed hindpaw (Figure 3 E, F).

Since the behavioral and paw swelling effects of CFA injection are present for at least 14 days, we used western blot to determine whether the GR downregulation in the L5 DRG was still present at this time point. As shown in Figure 4, 14 days after inflammation the GR expression was still significantly reduced compared to L5 DRGs from control rats.

Figure 4: Effect of local paw inflammation on GR expression in the DRG after 14 days of local paw inflammation.

Figure 4:

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Ipsilateral L5 DRGs were isolated from complete Freund’s adjuvant (CFA) injected rats 14 days after inflammation and compared to those from normal rats using Western blot analysis. A) Examples of observed bands. B) Quantification of GR protein showed reduction of the GR protein in the L5 DRG 14 days after CFA injection compared to L5 DRGs from normal rats. * p<0.05, significant difference between the groups (unpaired student t- test, p=0.0358). The results shown combine equal number of both sexes (n= 3 males and 3 females per group).

Some indices of chronic HPA axis activation were observed on POD14.

The decrease in GR in the “off-target” DRGs not directly innervating the inflamed paw was unexpected, and suggested some systemic factor might be responsible. One obvious candidate is elevation of plasma CORT levels, for example due to the stress of the pain model, which could reduce GR by the process of homologous downregulation6. We obtained blood samples for analysis of plasma CORT on POD1 and POD14, at 8:00 a.m. (i.e., when the circadian rhythm is near its nadir), close to the time (9:00 a.m.) at which animals in other experiments were sacrificed to obtain tissues for the immunohistochemical and western blot experiments. Males and females were analyzed separately due to the well-known sex effects on CORT levels18, 19. Two-way repeated measures ANOVA of the CORT concentration data for these 2 time points showed no overall effect of CFA, but an overall effect of time, in both males and females. However, post-hoc tests indicated that CORT was higher on POD14 compared to POD1 in both the males and in the females, only in the CFA-injected groups (Figure 5 A, B).

Figure 5. Effect of CFA injection on the HPA axis.

Figure 5.

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Several indicators of hypothalamus-pituitary-adrenal (HPA) axis were examined. A, B: Blood samples were obtained at 8:00 a.m., 1 and 14 days after complete Freund’s adjuvant (CFA) injection, and the plasma concentration of corticosterone (CORT) measured. Males (A) and females (B) were analyzed separately. **, p<0.01; ***, p<0.001, significant difference between the indicated values, Bonferroni posttest after 2-way repeated measures ANOVA. The overall effect of time was significant (males: p=0.004, F(1,22)=1.61; females, p =0.001, F(1,22) = 17.3 ); overall effect of CFA injection was not significant (males: p = 0.22, F(1,22) = 1.614; females, p =0.996, F(1,22 )= 0.00002), and interaction of time and CFA injection was not significant (males: p = 0.28, F(1,22) = 1.22; females, p = 0.149, F(1,22) = 0.149). After obtaining the POD 14 blood sample, rats were weighed and sacrificed and the heart, thymus, and adrenals isolated and weighed. Organ weights have been normalized to total body weight and expressed as a percent. *, p<0.05; n.s., not significantly different, unpaired t-test. Exact p values for comparisons (male, female): C: 0.0257, 0.200; D: 0.024, 0.1688; E: 0.3100, 0.0303; F: 0.4461, 0.6932. 3 outliers were removed based on the Grubb’s test: one female control adrenal weight, one male CFA heart weight, one male control heart weight. N = 12 rats per group. One female in the CFA group was missing the day CORT 1 measurement. All data passed normality test (Shapiro-Wilk) and equal variance test (Brown-Forsythe). Statistical comparisons between the 2 sexes were not made as these differences are well established in the literature.

Immediately after the POD14 blood sample was taken, animals were sacrificed and the adrenal, thymus, and heart dissected out and weighed. Organ weights were normalized to total body weight obtained just prior to sacrifice. As shown in Figure 5, some indications of chronic HPA activation and stress were obtained, which differed by sex: males with CFA injection demonstrated adrenal hypertrophy and decreased body weight, while females showed evidence of thymus involution. As a control, heart weight was not affected by CFA injection in either sex.

Microsympathectomy did not change the GR levels in the L5 DRG one day after CFA paw inflammation

We previously showed that a very localized microsympathectomy (“mSYMPX”), i.e. cutting the grey rami at the level of the L4 and L5 spinal nerves, reduced both behavioral sensitivity and paw swelling in the CFA model20. We therefore wanted to investigate if mSYMPX affects the GR levels in the DRG, which might contribute to these effects. We used western blot analysis to examine the GR protein levels one day after the CFA inflammation in the L5 DRG performed after 14 days of mSYMPX or sham mSYMPX (the same protocol as used in the previous study). GR expression in the L5 DRG one day after CFA inflammation was not significantly affected by the mSYMPX procedure (Supplemental File 3, Supplemental Figure 2 A, B).

Discussion

CFA hindpaw inflammation leads to pain and local edema. Immunohistochemical staining revealed that GR is widely expressed in normal DRG and skin tissues1–5, 21. CFA paw injection caused upregulation of GR in the skin on POD1, possibly reflecting the large influx of immune cells. In contrast, in the DRG, paw inflammation significantly reduced GR immunoreactivity on POD1. Western blotting confirmed this finding, and showed that this downregulation was maintained at POD 14. Interestingly, even though we used a very low dose of CFA and there were no signs of inflammation in the contralateral paw, GR protein expression was reduced in the contralateral L5 DRG at POD1, as well as in the ipsilateral L2 DRG, which does not innervate the paw. These data suggest that the CFA effect on GR levels in the DRG is not simply mediated by local inflammation of the peripheral fiber endings in the inflamed paw. Thus there are distinctive patterns of GR activation under different pain conditions and at different anatomical locations, which may have a significant impact on steroid treatments in these conditions. The GR is highly expressed in DRG neurons where it may serve to reduce hyperexcitability22, 23, so this downregulation might be expected to enhance pain behaviors and make them less responsive to steroid treatment.

The GR is downregulated by its own ligand, a process termed “homologous downregulation”, though many other local and systemic factors can also affect its expression6. CFA injection could in principle elevate plasma CORT levels, either directly through local production of pro-inflammatory cytokines that via the circulation directly increase pituitary adrenocorticotropic hormone (ACTH) release24–26, or because the induced pain acts as a stressor, stimulating ACTH release through higher brain circuitry27. Elevated plasma CORT seems unlikely to account for the observed decreases in GR expression observed in L5 and “off-target” DRGs on POD1, since the CORT levels were not affected by CFA in either sex on POD1. Plasma CORT samples were obtained at a time of day similar to the time at which samples were obtained (in different animals) for western blot or microscopy; namely in the morning, when in rodents the CORT levels are near their circadian nadir. The levels of GR protein fluctuate with a circadian rhythm following the CORT rhythm (e.g. in muscle28). HPA axis activity varies markedly throughout the circadian rhythm, with both basal (non-stress) plasma CORT, and the extent of post-stress CORT responses, varying with time of day29. As such, we cannot rule out the possibility that differences in plasma CORT levels at earlier points in the circadian cycle during the first 24 hours after CFA injection were integrated into differing GR levels in the DRG measured on POD1.

GR downregulation in the L5 DRG was also observed on POD14. We observed some indications of chronic HPA-axis activation at POD14, although these were not all robustly observed in both sexes. The increase in plasma CORT from day 1 to day 14 did not show an overall effect of CFA, although the post-tests showed a significant increase in only the CFA group. Other signs of chronic HPA activation and stress were observed only in one sex (females: 13% reduction in thymus weight; males: 29% increase in adrenal weight; males, 6% lower body weight). For comparison, in an earlier study, 30 days of chronic variable stress caused a 40% increase in adrenal weight and 18% decrease in thymus weight in male rats30. Thus it is plausible that chronic CORT elevation contributed to the observed downregulation of GR observed on POD14 in the L5 DRG, though other local factors may also be involved. A study in which the same volume and concentration of CFA that we used was injected into muscle, was unable to detect plasma elevation of several pro-inflammatory cytokines on POD1 despite detecting changes in the injection site of up to 10,00-fold in these cytokines31. Thus we deem it unlikely that the CFA injection model we used could elevate CORT levels by increasing systemic cytokines, and more likely that pain acting as a stressor could be a mechanism.

Our results differ from those of Li et al.5, who found upregulation of the GR in the DRG 4 days after CFA injection using both immunohistochemistry and western blot. The time-of-day for obtaining the samples was not given; one possibility is that the circadian fluctuation in corticosterone is altered by the CFA model and that time-of-day affects the result. They also reported no change in plasma CORT levels at this time point; however, these were from isoflurane anesthetized animals rather than morning non-stress samples as measured here. Our HPA axis studies also contrast with those of Shi et al.32, who found no effect of (a somewhat larger dose of) CFA on body weight, adrenal weight or plasma CORT (time of day not given) at 6 weeks after CFA injection, a longer time point than we evaluated here. In a mouse study using the CFA model, plasma CORT elevations were observed, but this dissipated by 12 hours despite pain behaviors lasting much longer33. Indices of HPA activation are seen in some other inflammatory pain models, such as the adjuvant-induced arthritis model27, 34.

Some such discrepancies between studies related to glucocorticoids and inflammatory pain could be related to differences between institutions and laboratories in stressors. Although acute stress can induce analgesia, under some circumstances stress can also enhance pain or unmask latent pain in animals that have apparently recovered from a mild pain model (e.g.35, 36). Some of these studies involve relatively minor stressors, making it plausible that small differences between different laboratories might affect the HPA axis and other stress-related systems, leading to discrepant results. Furthermore, until recently many such studies focused entirely on male animals. Important strain differences may also affect HPA axis reactivity33. While discrepant studies are unsettling, a similar variability is also observed in e.g. studies of steroid effects in human pain conditions37; if such factors can be systematically investigated new insights into the role of the GR in pain conditions may emerge.

Supplementary Material

Supplemental file 1

Supplemental file 1: gives additional details about experimental methods, along with Supplemental Table 1, showing animal numbers and disposition.

Supplemental file 2

Supplemental file 2: Supplemental Figure 1, showing confirmation of behavioral and paw swelling effects of the CFA model.

Supplemental file 3

Supplemental file 3: Supplemental Figure 2, showing effect of microsympathectomy on GR expression levels on POD1 of the CFA model.

Key Points Summary.

Question: How are glucocorticoid receptors regulated during peripheral inflammation?

Findings: The receptors are upregulated at the site of inflammation, but downregulated in the sensory ganglia including those contralateral to or not innervating the inflammation site.

Meaning: Downregulation of the glucocorticoid receptors in the sensory ganglia during peripheral inflammation may compromise the effectiveness of corticosteroids clinically used in inflammatory conditions, and modify the effects of endogenous glucocorticoids on the pain state.

Acknowledgements

This work was supported by in part by grants from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR068989 to J-M Zhang) and National Institute of Neurological Disorders and Stroke (NS045594, NS055860 to J-M Zhang), Bethesda, MD, USA. The authors thank Dana Busing for assistance with the corticosterone assay.

Glossary

ACTH

Adrenocorticotropic hormone

ANOVA

analysis of variance

CFA

Complete Freund’s adjuvant

CORT

corticosterone

DRG

dorsal root ganglion

EDTA

ethylenediaminetetraacetic acid

GAPDH

glyceraldehyde 3 phosphate dehydrogenase

GR

glucocorticoid receptor

HRP

Horse radish peroxidase

IHC

immunohistochemistry

mSYMPX

microsympathectomy

n.s.

not significant

PBST

phosphate-buffered saline with 0.3% Triton X-100

POD

postoperative day

RIA

radioimmunoassay

TBST

Tris buffered saline with Tween® 20

Footnotes

Conflicts of interest: none

References

  • 1.Lerch JK, Alexander JK, Madalena KM, et al. Stress Increases Peripheral Axon Growth and Regeneration through Glucocorticoid Receptor-Dependent Transcriptional Programs. eNeuro. Jul-Aug 2017;4(4). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Ibrahim SIA, Xie W, Strong JA, Tonello R, Berta T, Zhang JM. Mineralocorticoid Antagonist Improves Glucocorticoid Receptor Signaling and Dexamethasone Analgesia in an Animal Model of Low Back Pain. Front Cell Neurosci. 2018;12:453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Rijsdijk M, Agalave NM, van Wijck AJM, et al. Effect of intrathecal glucocorticoids on the central glucocorticoid receptor in a rat nerve ligation model. Scand J Pain. July 2017;16:1–9. [DOI] [PubMed] [Google Scholar]
  • 4.DeLeon M, Covenas R, Chadi G, Narvaez JA, Fuxe K, Cintra A. Subpopulations of primary sensory neurons show coexistence of neuropeptides and glucocorticoid receptors in the rat spinal and trigeminal ganglia. Brain Res. February 14 1994;636(2):338–342. [DOI] [PubMed] [Google Scholar]
  • 5.Li X, Shaqura M, Mohamed D, et al. Pro- versus Antinociceptive Nongenomic Effects of Neuronal Mineralocorticoid versus Glucocorticoid Receptors during Rat Hind Paw Inflammation. Anesthesiology. January 22 2018;128(796):809. [DOI] [PubMed] [Google Scholar]
  • 6.Dejager L, Vandevyver S, Petta I, Libert C. Dominance of the strongest: inflammatory cytokines versus glucocorticoids. Cytokine Growth Factor Rev. February 2014;25(1):21–33. [DOI] [PubMed] [Google Scholar]
  • 7.Almarestani L, Fitzcharles MA, Bennett GJ, Ribeiro-da-Silva A. Imaging studies in Freund’s complete adjuvant model of regional polyarthritis, a model suitable for the study of pain mechanisms, in the rat. Arthritis Rheum. June 2011;63(6):1573–1581. [DOI] [PubMed] [Google Scholar]
  • 8.Donaldson LF, Seckl JR, McQueen DS. A discrete adjuvant-induced monoarthritis in the rat: effects of adjuvant dose. J Neurosci Methods. August 1993;49(1–2):5–10. [DOI] [PubMed] [Google Scholar]
  • 9.Dong F, Xie W, Strong JA, Zhang J-M. Mineralocorticoid receptor blocker eplerenone reduces pain behaviors in vivo and decreases excitability in small diameter sensory neurons from local inflamed dorsal root ganglia in vitro. Anesthesiology. 2012;117(5):1102–1112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Sarabdjitsingh RA, Meijer OC, de Kloet ER. Specificity of glucocorticoid receptor primary antibodies for analysis of receptor localization patterns in cultured cells and rat hippocampus. Brain Res. May 17 2010;1331:1–11. [DOI] [PubMed] [Google Scholar]
  • 11.McKlveen JM, Myers B, Flak JN, et al. Role of prefrontal cortex glucocorticoid receptors in stress and emotion. Biol Psychiatry. November 1 2013;74(9):672–679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Boyle MP, Kolber BJ, Vogt SK, Wozniak DF, Muglia LJ. Forebrain glucocorticoid receptors modulate anxiety-associated locomotor activation and adrenal responsiveness. J Neurosci. February 15 2006;26(7):1971–1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Boyle MP, Brewer JA, Funatsu M, et al. Acquired deficit of forebrain glucocorticoid receptor produces depression-like changes in adrenal axis regulation and behavior. Proc Natl Acad Sci U S A. January 11 2005;102(2):473–478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Ulrich-Lai YM, Figueiredo HF, Ostrander MM, Choi DC, Engeland WC, Herman JP. Chronic stress induces adrenal hyperplasia and hypertrophy in a subregion-specific manner. Am J Physiol Endocrinol Metab. November 2006;291(5):E965–973. [DOI] [PubMed] [Google Scholar]
  • 15.Paskitti ME, McCreary BJ, Herman JP. Stress regulation of adrenocorticosteroid receptor gene transcription and mRNA expression in rat hippocampus: time-course analysis. Brain Res Mol Brain Res. September 15 2000;80(2):142–152. [DOI] [PubMed] [Google Scholar]
  • 16.Blanchard RJ, Nikulina JN, Sakai RR, McKittrick C, McEwen B, Blanchard DC. Behavioral and endocrine change following chronic predatory stress. Physiol Behav. February 15 1998;63(4):561–569. [DOI] [PubMed] [Google Scholar]
  • 17.Ulrich-Lai YM, Engeland WC. Hyperinnervation during adrenal regeneration influences the rate of functional recovery. Neuroendocrinology. February 2000;71(2):107–123. [DOI] [PubMed] [Google Scholar]
  • 18.Goel N, Workman JL, Lee TT, Innala L, Viau V. Sex differences in the HPA axis. Compr Physiol. July 2014;4(3):1121–1155. [DOI] [PubMed] [Google Scholar]
  • 19.Critchlow V, Liebelt RA, Bar-Sela M, Mountcastle W, Lipscomb HS. Sex difference in resting pituitary-adrenal function in the rat. Am J Physiol. November 1963;205(5):807–815. [DOI] [PubMed] [Google Scholar]
  • 20.Xie W, Chen S, Strong JA, Li A-L, Lewkowich IP, Zhang J-M. Localized sympathectomy reduces mechanical hypersensitivity by restoring normal immune homeostasis in rat models of inflammatory pain. J Neurosci. 2016;36(33):8712–8725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Shaqura M, Li X, Al-Khrasani M, et al. Membrane-bound glucocorticoid receptors on distinct nociceptive neurons as potential targets for pain control through rapid non-genomic effects. Neuropharmacology. December 2016;111:1–13. [DOI] [PubMed] [Google Scholar]
  • 22.Ye L, Xie W, Strong JA, Zhang JM. Blocking the mineralocorticoid receptor improves effectiveness of steroid treatment for low back pain in rats. Anesthesiology. April 28 2014;121(3):632–643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Breivik H Local Anesthetic Blocks and Epidurals In: McMahon S, Koltzenburg M, Tracey I, Turk D, eds. Wall and Mekzack’s Textbook of Pain. 6th ed. Philadelphia, PA: Saunders Elsevier; 2013:523–537. [Google Scholar]
  • 24.Tracey KJ. The inflammatory reflex. Nature. December 19–26 2002;420(6917):853–859. [DOI] [PubMed] [Google Scholar]
  • 25.Serrats J, Grigoleit JS, Alvarez-Salas E, Sawchenko PE. Pro-inflammatory immune-to-brain signaling is involved in neuroendocrine responses to acute emotional stress. Brain Behav Immun. May 2017;62:53–63. [DOI] [PubMed] [Google Scholar]
  • 26.Serrats J, Schiltz JC, Garcia-Bueno B, van Rooijen N, Reyes TM, Sawchenko PE. Dual roles for perivascular macrophages in immune-to-brain signaling. Neuron. January 14 2010;65(1):94–106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Bomholt SF, Harbuz MS, Blackburn-Munro G, Blackburn-Munro RE. Involvement and role of the hypothalamo-pituitary-adrenal (HPA) stress axis in animal models of chronic pain and inflammation. Stress. March 2004;7(1):1–14. [DOI] [PubMed] [Google Scholar]
  • 28.Yao Z, DuBois DC, Almon RR, Jusko WJ. Modeling circadian rhythms of glucocorticoid receptor and glutamine synthetase expression in rat skeletal muscle. Pharm Res. April 2006;23(4):670–679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Spencer RL, Chun LE, Hartsock MJ, Woodruff ER. Glucocorticoid hormones are both a major circadian signal and major stress signal: How this shared signal contributes to a dynamic relationship between the circadian and stress systems. Front Neuroendocrinol. April 2018;49:52–71. [DOI] [PubMed] [Google Scholar]
  • 30.Herman JP, Adams D, Prewitt C. Regulatory changes in neuroendocrine stress-integrative circuitry produced by a variable stress paradigm. Neuroendocrinology. February 1995;61(2):180–190. [DOI] [PubMed] [Google Scholar]
  • 31.Niu KY, Ro JY. Changes in intramuscular cytokine levels during masseter inflammation in male and female rats. Neurosci Lett. January 7 2011;487(2):223–227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Shi M, Wang JY, Luo F. Depression shows divergent effects on evoked and spontaneous pain behaviors in rats. J Pain. March 2010;11(3):219–229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Benedetti M, Merino R, Kusuda R, et al. Plasma corticosterone levels in mouse models of pain. Eur J Pain. July 2012;16(6):803–815. [DOI] [PubMed] [Google Scholar]
  • 34.Windle RJ, Wood SA, Kershaw YM, Lightman SL, Ingram CD, Harbuz MS. Increased corticosterone pulse frequency during adjuvant-induced arthritis and its relationship to alterations in stress responsiveness. J Neuroendocrinol. October 2001;13(10):905–911. [DOI] [PubMed] [Google Scholar]
  • 35.Rivat C, Laboureyras E, Laulin JP, Le Roy C, Richebe P, Simonnet G. Non-nociceptive environmental stress induces hyperalgesia, not analgesia, in pain and opioid-experienced rats. Neuropsychopharmacology. October 2007;32(10):2217–2228. [DOI] [PubMed] [Google Scholar]
  • 36.Marvizon JC, Walwyn W, Minasyan A, Chen W, Taylor BK. Latent sensitization: a model for stress-sensitive chronic pain. Curr Protoc Neurosci. April 1 2015;71:9 50 51–14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Deer TR, Grider JS, Pope JE, et al. The MIST Guidelines: The Lumbar Spinal Stenosis Consensus Group Guidelines for Minimally Invasive Spine Treatment. Pain Pract. March 2019;19(3):250–274. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental file 1

Supplemental file 1: gives additional details about experimental methods, along with Supplemental Table 1, showing animal numbers and disposition.

NIHMS1588906-supplement-Supplemental_file_1.pdf (693.6KB, pdf)
Supplemental file 2

Supplemental file 2: Supplemental Figure 1, showing confirmation of behavioral and paw swelling effects of the CFA model.

NIHMS1588906-supplement-Supplemental_file_2.pdf (190.2KB, pdf)
Supplemental file 3

Supplemental file 3: Supplemental Figure 2, showing effect of microsympathectomy on GR expression levels on POD1 of the CFA model.

NIHMS1588906-supplement-Supplemental_file_3.pdf (446.9KB, pdf)

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