Isoflurane stress induces glucocorticoid production in mouse lymphoid organs

in Journal of Endocrinology
Authors:
Jordan E HamdenDepartment of Zoology, University of British Columbia, Vancouver, British Columbia, Canada
Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, British Columbia, Canada

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Melody SalehzadehDepartment of Zoology, University of British Columbia, Vancouver, British Columbia, Canada
Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, British Columbia, Canada

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Katherine M GrayDepartment of Psychology, University of British Columbia, Vancouver, British Columbia, Canada
Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada

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Brandon J ForysDjavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, British Columbia, Canada
Department of Psychology, University of British Columbia, Vancouver, British Columbia, Canada

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Kiran K SomaDepartment of Zoology, University of British Columbia, Vancouver, British Columbia, Canada
Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, British Columbia, Canada
Department of Psychology, University of British Columbia, Vancouver, British Columbia, Canada
Graduate Program in Neuroscience, University of British Columbia, Vancouver, British Columbia, Canada

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Correspondence should be addressed to K K Soma: ksoma@psych.ubc.ca
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Glucocorticoids (GCs) are secreted by the adrenal glands and locally produced by lymphoid organs. Adrenal GC secretion at baseline and in response to stressors is greatly reduced during the stress hyporesponsive period (SHRP) in neonatal mice (postnatal day (PND) 2–12). It is unknown whether lymphoid GC production increases in response to stressors during the SHRP. Here, we administered an acute stressor (isoflurane anesthesia) to mice before, during, and after the SHRP and measured systemic and local GCs via mass spectrometry. We administered isoflurane, vehicle control (oxygen), or neither (baseline) at PND 1, 5, 9, or 13 and measured progesterone and six GCs in blood, bone marrow, thymus, and spleen. At PND1, blood and lymphoid GC levels were high and did not respond to stress. At PND5, blood GC levels were very low and increased slightly after stress, while lymphoid GC levels were also low but increased greatly after stress. At PND9, blood and lymphoid GC levels were similar at baseline and increased similarly after stress. At PND13, blood GC levels were higher than lymphoid GC levels at baseline, and blood GC levels showed a greater response to stress. These data demonstrate the remarkable plasticity of GC physiology during the postnatal period, show that local steroid levels do not reflect systemic steroid levels, provide insight into the SHRP, and identify a potential mechanism by which early-life stressors can alter immunity in adulthood.

Abstract

Glucocorticoids (GCs) are secreted by the adrenal glands and locally produced by lymphoid organs. Adrenal GC secretion at baseline and in response to stressors is greatly reduced during the stress hyporesponsive period (SHRP) in neonatal mice (postnatal day (PND) 2–12). It is unknown whether lymphoid GC production increases in response to stressors during the SHRP. Here, we administered an acute stressor (isoflurane anesthesia) to mice before, during, and after the SHRP and measured systemic and local GCs via mass spectrometry. We administered isoflurane, vehicle control (oxygen), or neither (baseline) at PND 1, 5, 9, or 13 and measured progesterone and six GCs in blood, bone marrow, thymus, and spleen. At PND1, blood and lymphoid GC levels were high and did not respond to stress. At PND5, blood GC levels were very low and increased slightly after stress, while lymphoid GC levels were also low but increased greatly after stress. At PND9, blood and lymphoid GC levels were similar at baseline and increased similarly after stress. At PND13, blood GC levels were higher than lymphoid GC levels at baseline, and blood GC levels showed a greater response to stress. These data demonstrate the remarkable plasticity of GC physiology during the postnatal period, show that local steroid levels do not reflect systemic steroid levels, provide insight into the SHRP, and identify a potential mechanism by which early-life stressors can alter immunity in adulthood.

Introduction

Glucocorticoids (GCs) are steroids produced by the adrenal glands and within lymphoid organs such as the bone marrow, thymus, and spleen (Schmidt et al. 2008, Taves et al. 2017). In mice, corticosterone is the primary active GC. Evidence for lymphoid production of GCs includes expression of steroidogenic enzymes, in vitro production of corticosterone, in vivo corticosterone measurement, and studies of knockout mouse models (Taves et al. 2015, 2016, Mittelstadt et al. 2018, Hamden et al. 2019). Within lymphoid tissues, corticosterone can be synthesized from cholesterol or other precursors or regenerated from the inactive metabolite 11-dehydrocorticosterone (DHC) (Taves et al. 2016) (Fig. 1). Importantly, the environmental factors that drive local corticosterone production are unknown, but might be of greater importance during early life, when adrenal GC production is low in altricial species, termed the stress hyporesponsive period (SHRP).

Figure 1
Figure 1

Simplified glucocorticoid synthetic pathway. Boxes indicate steroids measured in this study.

Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0154

The SHRP is characterized by very low circulating corticosterone levels at baseline and relatively small increases in circulating corticosterone levels in response to stressors (Henning 1978, Meaney et al. 1985, D’Amato et al. 1992, Schmidt et al. 2002, 2003). The SHRP in mice ranges from approximately postnatal day 2 (PND2) to PND12 (with PND0 defined as the day of birth) (Schmidt et al. 2003). There is a similar developmental period in humans, from ~4 months to ~5 years (Gunnar & Donzella 2002). Studies of the SHRP in mice and rats demonstrate that some stressors such as ether, shock, cold, and novelty produce little to no increase in circulating corticosterone levels, while other stressors such as endotoxin and maternal separation (≥4 h) produce slightly larger increases in circulating cortisterone levels (Haltmeyer et al. 1966, Gray 1971, Butte et al. 1973, Cote & Yasumura 1975, Tang & Phillips 1977, Walker et al. 1990, 1991, D’Amato et al. 1992, Cirulli et al. 1994, 1997, del Rey et al. 1996, Spinedi et al. 1997, Schmidt et al. 2002, 2003). The purpose of the SHRP is not well understood but may be to protect rapidly developing tissues from deleterious effects of high GC levels (Sapolsky & Meaney 1986). Interestingly, during the SHRP, GC levels are higher in lymphoid organs than in blood, suggesting local GC production may facilitate lymphocyte development (Taves et al. 2015, Hamden et al. 2019).

During chronic stress, high levels of corticosterone are prolonged and highly immunosuppressive, but corticosterone is also a modulator of lymphocyte development when levels are moderate or low. Corticosterone is necessary for a competent T cell repertoire and modulates T cell development by altering T cell selection (Lu et al. 2000, Mittelstadt et al. 2012, 2018, Taves & Ashwell 2020). Increased GC signaling leads to the positive selection of more strongly reactive T cell antigen receptors (Iwata et al. 1991, Taves et al. 2019). Further, increased GC signaling results in more T cells surviving negative selection (Vacchio et al. 1994, 1999, Vacchio & Ashwell 1997). Together, these data clearly demonstrate that increased corticosterone signaling modulates thymocyte development by shifting the window for positive selection in favor of more reactive T cells and allowing more cells to survive negative selection (Van Laethem et al. 2001). Less is known about the role of corticosterone in B cell development. Developing B cells express glucocorticoid receptor (GR) and undergo GC-induced apoptosis (Garvy et al. 1993, Trottier et al. 2008). Chronic elevation of GCs reduces all B cell subset populations in the bone marrow (Gruver-Yates et al. 2014, Taves et al. 2016). GCs also inhibit B cell receptor (BCR) signaling in leukemia cells in vitro, suggesting a role for GCs in BCR function similar to their role in T cell receptor selection (Cortez et al. 1996, Cain et al. 2020).

Endotoxin exposure and maternal separation during the SHRP have long-lasting programming effects on the immune system, referred to as perinatal programming. These stressors produce widespread changes in the immune system and hypothalamic–pituitary–adrenal axis, usually seen after a second 'hit' in adulthood, deemed the 'two-hit model' (Shanks et al. 2000, Hodgson et al. 2001, Bilbo et al. 2005, Avitsur & Sheridan 2009, Walker et al. 2010). The effects of other stressors such as ether, shock, cold, and novelty on perinatal programming are less studied. This is likely due to the lack of increases in systemic corticosterone levels associated with these stressors and thus a seemingly limited ability to impact development. However, local production of corticosterone within lymphoid tissues may allow tissues to increase local levels in response to stressors and permit seemingly mild stressors to have strong perinatal programming effects.

To determine whether a stressor administered before, during, or after the SHRP induces local corticosterone production in lymphoid organs, we treated mice at four ages with isoflurane (stressor), vehicle control (oxygen), or neither (baseline). Isoflurane is an inhalable anesthetic and a halogenated ether, similar to anesthetics previously used for SHRP studies (Gray 1971, Butte et al. 1972, Tang & Phillips 1977). We measured a panel of seven steroids in the blood, bone marrow, thymus, and spleen by liquid chromatography tandem mass spectrometry (LC-MS/MS). By collecting data on corticosterone, its precursors, and metabolite in the blood and lymphoid organs, we can better understand the contributions of adrenally produced and locally produced GCs to local GC levels before and after stress.

Materials and methods

Subjects

C57BL/6J breeding mouse pairs (n = 12) were housed in a specific pathogen-free colony in the Centre for Disease Modeling at the University of British Columbia. Mouse pups were housed undisturbed in their home cage with both parents until treatment. Cages were checked for new litters twice a day, and litter sizes ranged from 5 to 11 animals. Colony rooms were maintained between 20 and 22°C with 40–70% relative humidity. Mice were housed in ventilated Ehret polysulfone Type IIL filter top cages, with beta-chip bedding, under a 14 h light:10 h darkness cycle (lights on 6:00–20:00 h), with free access to water (purified by reverse osmosis and sterilized by chlorination) and food (Teklad Diet 2919). A red translucent hut and nestlet were placed in each cage for enrichment. All procedures complied with the Canadian Council on Animal Care, and protocols were approved by the University of British Columbia Animal Care Committee.

Isoflurane treatment and tissue collection

On the first day that pups were present in the cage (PND0), pups were randomly assigned to a treatment (baseline, oxygen, or isoflurane) and experimental age (PND1, 5, 9, or 13) (n = 10 pups/treatment/age). The oxygen group was included as a vehicle control. Each treatment group at a particular age included two to three pups from the same litter and incorporated at least four litters from different breeding pairs. PND1 (pre-SHRP), PND5 (early-SHRP), PND9 (late-SHRP), and PND13 (post-SHRP) were selected to provide ages before, during, and after the SHRP in mice (Schmidt et al. 2003). Both systemic and local GC levels have been shown to change dramatically across this early developmental period (Schmidt et al. 2003, Taves et al. 2015, Hamden et al. 2019). All experiments were conducted between 9:00 and 11:00 h to reduce diurnal variation in steroid levels.

On the day of treatment, animals assigned to the baseline group were euthanized by rapid decapitation, without the use of anesthetic, less than 1 min after initial disturbance of the cage. Animals assigned to the oxygen or isoflurane treatments were placed in an induction chamber with a small amount of nesting material from their home cage and a heating pad under the chamber. For the oxygen treatment, oxygen was turned on at 2 L/min prior to placing animals in the induction chamber. Animals were left in the chamber for 30 min and euthanized by rapid decapitation without the use of isoflurane. For the isoflurane treatment, oxygen was turned on at 2 L/min with 5% isoflurane prior to placing animals in the induction chamber. After 3 min of exposure, isoflurane was turned off, and animals were left in the chamber with oxygen for 27 min (total time 30 min) and euthanized by rapid decapitation without the use of further anesthetic. The 3-min isoflurane exposure is similar to previous studies using ether (Gray 1971, Butte et al. 1972, Tang & Phillips 1977), and 30 min is a common endpoint in neonatal stress studies and when circulating corticosterone levels peak (Gray 1971, D’Amato et al. 1992, Schmidt et al. 2002, 2003). Whole blood (hereafter 'blood'), femurs, thymus, and spleen were collected and immediately frozen on dry ice and stored at −70ºC until steroid extraction. Sex of mice was determined by the University of British Columbia Genotyping Facility using PCR of tail clips.

Steroid analysis by LC-MS/MS

Steroids were extracted from the blood, femur, bone marrow, thymus, and spleen via liquid–liquid extraction and analyzed by LC-MS/MS as before (Hamden et al. 2021) with slight modification. The limit of detection for progesterone, 11-deoxycorticosterone (DOC), corticosterone, DHC, 11-deoxycortisol, cortisol, and cortisone was improved four-fold, from 0.2 to 0.05 pg. Standard curves, controls, blanks (solvent with deuterated internal standard), and double blanks (solvent only) were extracted and analyzed alongside all samples. Pooled mouse serum was used as an inter-assay control (n = 3/assay) to allow for comparison across all assays used in this study. A steroid was considered non-detectable if the quantifier and qualifier transitions were not present.

Statistical analysis

For groups with less than 40% non-detectable measurements, the data for non-detectable samples were imputed (Wei et al. 2018, Tobiansky et al. 2020). Data were imputed for each age, treatment, and steroid independently. At least 60% of measurements for progesterone, DOC, corticosterone, and DHC were detectable in each group and in 100, 91, 100, and 95% of total samples, respectively. In contrast, 11-deoxycortisol, cortisol, and cortisone were non-detectable in all samples. To make comparisons between blood and tissues, 1 mL of blood was considered to weigh 1 g, as before (Schmidt & Soma 2008, Taves et al. 2011, 2015, Hamden et al. 2019). We weighed 50, 20, and 5 μL of blood using an ultrasensitive balance (n = 5/volume). These volumes of blood weighed (average ±s.e.m.) 51.9 ± 0.4, 20.9 ± 0.2, and 5.3 ± 0.03 mg, respectively. These data indicate that 1 mL of blood weighs 1.05 ± 0.004 g.

There was no effect of sex in baseline or stressed steroid levels and no interaction between sex and any other factor (P ≥ 0.83, in all cases) in these pre-pubertal animals. Thus, data from both sexes were pooled for further analysis, as before (Spinedi et al. 1997, McCormick et al. 1998, Taves et al. 2015, Hamden et al. 2021).

Data were analyzed in three complementary ways. First, data were analyzed for an effect of treatment and tissue for each steroid separately at each age by mixed-effects model (α = 0.05 for all statistical tests). When there was a significant main effect of treatment or a tissue × treatment interaction, post hoc analyses were performed by Tukey’s multiple comparison test to determine differences among treatment groups within each tissue. Second, to compare the difference between blood and lymphoid steroid levels across treatment and age, blood steroid values were subtracted from tissue steroid values. These difference scores were analyzed for an effect of treatment and age by two-way ANOVA followed by Tukey’s multiple comparison test. Third, multiple linear regression was used to elucidate which systemic and local steroids were most important in predicting local corticosterone levels at each age and treatment. To ensure homogeneity of variance, data were log transformed prior to analysis when necessary. For multiple linear regression, steroid levels were converted to molarity to allow comparisons across steroids. All graphs are presented using non-transformed data as average ±s.e.m.. All data were analyzed using GraphPad Prism 9 (version 9.0.0) and R (version 4.0.2 'Taking Off Again').

Results

Steroid levels

PND1

For progesterone, DOC, corticosterone, and DHC, there were main effects of tissue (P ≤ 0.02 in all cases) but no main effects of treatment or tissue × treatment interactions (Fig. 2). Steroid levels were higher in bone marrow, thymus, and spleen than in blood. Corticosterone levels were higher than those of other steroids.

Figure 2
Figure 2

In post-natal day 1 (PND1) mice, concentrations of (A) progesterone, (B) 11-deoxycorticosterone (DOC), (C) corticosterone, and (D) 11-dehydrocorticosterone (DHC) in whole blood, bone marrow, thymus, and spleen. Data are shown as mean ± s.e.m. There was no significant main effect of treatment or a tissue × treatment interaction, and thus, nopost hoc test was performed. n = 10 for all steroids and tissues. A full color version of this figure is available at https://doi.org/10.1530/JOE-21-0154.

Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0154

PND5

For progesterone, DOC, corticosterone, and DHC, there were main effects of tissue (P≤ 0.0001 in all cases), main effects of treatment (P  < 0.01 in all cases), and tissue × treatment interactions (P  < 0.0001 in all cases) (Fig. 3). In all groups, progesterone levels were higher in all tissues than in blood. For DOC, there was a significant increase in all tissues after isoflurane treatement compared to baseline. For corticosterone and DHC, there was a significant increase in all tissues after oxygen and isoflurane treatements compared to baseline. DOC, corticosterone, and DHC levels increased more in bone marrow, thymus, and spleen than in blood (Fig. 3 and Supplementary Table 1, see section on supplementary materials given at the end of this article). Corticosterone levels were higher than those of other steroids. Increases in DOC, corticosterone, and DHC levels after isoflurane treatment were far greater in the lymphoid organs than in blood (Supplementary Fig. 3). In general, steroid levels were lower in blood than in lymphoid organs.

Figure 3
Figure 3

In post-natal day 5 (PND5) mice, concentrations of (A) progesterone, (B) 11-deoxycorticosterone (DOC), (C) corticosterone, and (D) 11-dehydrocorticosterone (DHC) in whole blood, bone marrow, thymus, and spleen. Data are shown as mean ± s.e.m. Tukey’s post hoc test was used to determine differences in baseline, oxygen, and isoflurane groups within each tissue, significant differences are denoted by letters. n = 10 for all steroids and tissues. For exact P values, see Supplementary Table 1. A full color version of this figure is available at https://doi.org/10.1530/JOE-21-0154.

Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0154

PND9

For progesterone and DHC, there were main effects of tissue (P  < 0.0001 in both cases), main effects of treatment (P ≤ 0.01 in both cases), and tissue × treatment interactions (P  < 0.0001 in both cases). For DOC and corticosterone, there were main effects of tissue (P  < 0.0001 in both cases) and main effects of treatment (P  < 0.0001 in both cases), but no tissue × treatment interactions (Fig. 4). For progesterone, levels increased after isoflurane treatment compared to baseline in blood, thymus, and spleen but not in bone marrow. For DOC, corticosterone, and DHC, there were significant increases in steroid levels after isoflurane treatment compared to baseline and oxygen, and the increases were similar across all tissues (Fig. 4 and Supplementary Table 1). Corticosterone levels were higher than those of other steroids. Increases in DOC, corticosterone, and DHC levels after isoflurane treatment were similar in blood and lymphoid organs. In general, steroid levels were similar between blood and lymphoid organs.

Figure 4
Figure 4

In post-natal day 9 (PND9) mice, concentrations of (A) progesterone, (B) 11-deoxycorticosterone (DOC), (C) corticosterone, and (D) 11-dehydrocorticosterone (DHC) in whole blood, bone marrow, thymus, and spleen. Data are shown as mean ± s.e.m. Tukey’s post hoc test was used to determine differences in baseline, oxygen, and isoflurane groups within each tissue, significant differences are denoted by letters. n = 10 for all steroids and tissues. For exact P values, see Supplementary Table 1. A full color version of this figure is available at https://doi.org/10.1530/JOE-21-0154.

Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0154

PND13

For progesterone, DOC, and DHC, there were main effects of tissue (P ≤ 0.04 in all cases), main effects of treatment (P  < 0.0001 in all cases), but no tissue × treatment interactions. For corticosterone, there was a main effect of tissue (P  < 0.0001), a main effect of treatment (P  < 0.0001), and a tissue × treatment interaction (P  = 0.04) (Fig. 5). For progesterone and DOC, there were significant increases after isoflurane treatment compared to baseline and oxygen treatments. For corticosterone and DHC, there were significant increases after isoflurane treatment compared to baseline, but there were no differences between oxygen and isoflurane treatments in any tissue (Fig. 5 and Supplementary Table 1). Corticosterone levels were higher than those of other steroids. Increases in DOC, corticosterone, and DHC levels after isoflurane treatment were greater in blood than in lymphoid organs. In general, steroid levels were higher in blood than in lymphoid organs.

Figure 5
Figure 5

In post-natal day 13 (PND13) mice, concentrations of (A) progesterone, (B) 11-deoxycorticosterone (DOC), (C) corticosterone, (D) 11-dehydrocorticosterone (DHC) in whole blood, bone marrow, thymus, spleen. Data are shown as mean ± s.e.m.. Tukey’s post hoc test was used to determine differences in baseline, oxygen, and isoflurane groups within each tissue, significant differences are denoted by letters. n = 10 for all steroids and tissues. For exact P values, see Supplementary Table 1. A full color version of this figure is available at https://doi.org/10.1530/JOE-21-0154.

Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0154

Difference in blood and lymphoid tissue corticosterone levels

To determine whether the relationship between blood and tissue steroid levels at baseline and after stress changes across age, we subtracted blood steroid levels from tissue steroid levels. For these difference scores, there was a main effect of treatment for bone marrow only (P  = 0.04), a main effect of age in all tissues (P  < 0.0001 in all cases), and a treatment × age interaction in all tissues (P  < 0.0001 in all cases) (Fig. 6 and Supplementary Table 3). There were no significant differences across ages in the baseline group for any tissue, and blood and lymphoid organ corticosterone levels were similar at all ages. Additionally, there were no significant differences across ages in the oxygen group for bone marrow and spleen; PND5 and PND9 were different from PND13 for thymus. Further, lymphoid organ and blood corticosterone levels were generally similar.

Figure 6
Figure 6

In post-natal day (PND) 5, 9, and 13 mice, difference in tissue – blood corticosterone levels in (A) bone marrow, (B) thymus, (C) spleen. Data are show as mean ± s.e.m.. Tukey’s post hoc test was used to determine differences between PND5, PND9, and PND13 corticosterone levels within each treatment group, significant differences are denoted by letters. n = 10 for all ages and tissues. For exact P values, see Supplementary Table 4. A full color version of this figure is available at https://doi.org/10.1530/JOE-21-0154.

Citation: Journal of Endocrinology 251, 2; 10.1530/JOE-21-0154

Predictors of local corticosterone levels

Using multiple linear regression, we were able to predict local corticosterone levels within lymphoid organs at each age. For each lymphoid organ, the best model included corticosterone levels in the blood as well as DOC and DHC levels in the blood and each lymphoid organ, respectively, but not progesterone levels in the blood or lymphoid organs. Results of the regression analysis and significant predictors are presented in Supplementary Table 4. Briefly, at PND1, we were able to predict local corticosterone levels in all tissues for all treatments (except baseline bone marrow). At PND5, we were able to predict local corticosterone levels in all tissues at baseline and in the spleen after oxygen. At PND9, we were able to predict local corticosterone levels in the thymus and spleen at baseline and in all tissues after oxygen. At PND13, we were able to predict corticosterone levels in all tissues at baseline, in the thymus and spleen after oxygen treatment, and in the spleen after isoflurane treatment. Overall, our model was effective at predicting local corticosterone levels at baseline and after oxygen treatments but less effective at predicting local levels after isoflurane treatment.

Discussion

The SHRP has long been thought to protect developing tissues from the deleterious effects of high GC levels. Emerging evidence on the local production of GCs during the SHRP may refine this idea by allowing tissues that require GCs for development to locally increase levels. These data provide further evidence to support this hypothesis by demonstrating that an acute stressor (isoflurane anesthesia) during neonatal development causes small, age-dependent increases in systemic corticosterone levels but causes large increases in local GC levels within the SHRP. At PND1, mice are non-responsive to isoflurane stress. Lymphoid GC levels were greater than blood GC levels at PND1 but did not increase with stress. Baseline tissue GC levels were similar to blood GC levels at PND5 and PND9 and lower than blood GC levels at PND13, but tissue levels increased with stress at all three ages. Counter to our results on blood GC levels, local GC levels peaked at PND5 and were similar at PND9 and PND13. Together, these data suggest that lymphoid organs increase local production of GCs during the SHRP in response to stress and that local production may be particularly important at PND5. Local GC production at PND5 has important implications for rodent models of early-life stress, as stressors are commonly administered at this age.

Systemic glucocorticoid levels

The SHRP has been characterized by ultra-low baseline corticosterone concentrations in the blood and relatively small increases in response to a variety of stressors (Cirulli et al. 1994, 1997, D’Amato et al. 1992, Spinedi et al. 1997, Schmidt et al. 2002, 2003). Here, we report very high corticosterone levels at PND1, as seen before in C57Bl/6J mice (Taves et al. 2015), but not in CD1 mice that have very low PND1 corticosterone levels (Schmidt et al. 2002). This might reflect a strain difference. Importantly, PND0 is defined as the day of birth in all three studies. Consistent with this study, neither strain showed an increase in corticosterone levels in response to stress (Schmidt et al. 2002). At PND1, the large variability in blood corticosterone levels might be explained by varying times after birth (Boksa 1997). At PND5, blood corticosterone levels were very low at baseline and showed a very small increase in response to isoflurane stress (<2 ng/mL increase). At PND9, blood corticosterone levels were still very low at baseline but had a greater increase in response to stress (~10 ng/mL) than on PND5. These data at PND5 and 9 are comparable to previous studies using ether, a similar inhalant anesthetic (Gray 1971, Tang & Phillips 1977, Schoenfeld et al. 1980). At PND13, blood corticosterone levels were higher at baseline (although lower than adult levels) (Hamden et al. 2019), and levels increased greatly after stress, consistent with the end of the SHRP (Schmidt et al. 2003). These data suggest that rather than developing in two distinct phases, the HPA axis may develop gradually, with full maturation occurring after PND13.

To our knowledge, these are the first data from animals in the SHRP on blood progesterone, DOC, and DHC levels after a stressor. Baseline blood progesterone levels were highest at PND1 and similar across the other ages, as previously reported (Taves et al. 2015). Progesterone levels responded as did corticosterone levels, with greater increases in response to stress in older animals. As expected, DOC and DHC levels followed the same developmental pattern as corticosterone levels. The highest levels were observed at PND1, the lowest levels at PND5, and levels increased similarly to corticosterone in response to stress. Baseline DOC and DHC levels at PND5 are in agreement with previous reports (Taves et al. 2015, Hamden et al. 2019, 2021). Together, these data show that circulating progesterone, DOC, corticosterone, and DHC levels follow a similar developmental pattern and that blood steroid levels increase minimally in response to stress during the SHRP. Thus, during the SHRP, progesterone, DOC, and corticosterone are all likely secreted from the adrenal.

Sex differences in blood GC levels are well known in adult rodents, with females having higher baseline levels and greater increases in response to stressors. In contrast, we found no sex differences in circulating GC levels at baseline or after stress in neonatal mice, as in previous work (Spinedi et al. 1997, McCormick et al. 1998, Taves et al. 2015, Hamden et al. 2019, 2021). Three studies also reported no effects of sex in older, but still pre-pubescent, mice (Taves et al. 2015, Hamden et al. 2019, 2021). These data suggest that sex differences in GC levels may be driven by an increase in gonadal sex steroids at puberty.

Local glucocorticoid levels

Similar to blood steroid levels, local steroid levels increased in response to isoflurane stress after PND1. However, after stress, lymphoid steroid levels were much higher than blood steroid levels at PND5, similar to blood steroid levels at PND9, and lower than blood steroid levels at PND13.

These data provide further evidence of local production of GCs within lymphoid organs, particularly at PND5. Previous studies demonstrated that neonatal lymphoid organs possess all the necessary synthetic and regenerative enzymes to produce corticosterone, produce corticosterone in vitro from precursors and DHC, and have locally elevated corticosterone levels at baseline in vivo (Vacchio et al. 1994, Taves et al. 2015, Mittelstadt et al. 2018). Here, we demonstrate that an acute stressor dramatically increases GC levels within lymphoid organs at PND5, despite a small increase in blood GC levels. Given the wealth of evidence that lymphoid organs produce GCs, these data suggest that isoflurane stress rapidly increases local corticosterone production in lymphoid organs.

It is unclear what factor(s) stimulates local GC production, but adrenocorticotropin hormone (ACTH) is a likely candidate. The SHRP is caused by a combination of high negative feedback, low circulating ACTH levels, and low adrenal responsiveness to ACTH (Schmidt 2019). Interestingly, lymphocytes in the bone marrow, thymus, and spleen express the ACTH receptor (MC2R), suggesting they respond to circulating ACTH (Clarke & Bost 1989, Johnson et al. 2001). These data raise the possibility that, during the neonatal period, circulating ACTH mediates the effects of isoflurane stress on lymphoid GC production. Such a mechanism would increase spatial specificity of GC signaling and prevent deleterious effects of high systemic GC levels on somatic growth and brain development during this critical period (Nishi et al. 2014).

Lymphoid organs can modulate local corticosterone levels by at least four mechanisms, which are not mutually exclusive. First, free corticosterone can passively diffuse into organs from the blood. Second, organs can locally synthesize corticosterone from cholesterol. Third, organs can locally metabolize corticosterone to DHC or regenerate corticosterone from local DHC. Fourth, DOC and DHC in the blood can diffuse into organs and be locally converted to corticosterone. All four of these mechanisms might contribute to the local corticosterone elevation in response to isoflurane stress. Increased lymphoid progesterone and DOC levels are likely a result of both diffusion from blood and de novo production from cholesterol, but levels may remain lower than lymphoid corticosterone levels because they are converted to the active steroid. Locally increased DHC levels are likely due to diffusion of DHC from blood and conversion of corticosterone to DHC by 11β-HSD2 activity. Similar to progesterone and DOC, local DHC levels may remain lower than local corticosterone levels because DHC can also be regenerated back into corticosterone by 11β-HSD1 activity or released into blood for clearance. Future studies can further elucidate the mechanism of local corticosterone production by treatment with inhibitors of Cyp11B1 or 11β-HSD1 activity. A build-up of precursors within lymphoid organs after treatment with inhibitor would indicate corticosterone synthesis, while a build-up of DHC would indicate the importance of regeneration in determining local corticosterone levels.

Alternatively, locally elevated corticosterone levels after stress could be due to sequestration rather than local production, but this is not likely. If corticosterone were not locally produced, we would expect that local GC levels would closely reflect blood GC levels, unless corticosterone is sequestered by binding to the GR, higher-affinity mineralocorticoid receptor (MR), or corticosteroid binding globulin within tissues. However, glucocorticoid binding of MR in lymphoid tissues is very low (Miller et al. 1990). Further, DOC and DHC are also higher within lymphoid organs than in blood, and DOC and DHC have lower binding affinity than corticosterone to GR and MR. Overall, the most likely explanation of locally increased DOC, corticosterone, and DHC is local production of GCs.

Contrary to previous reports (Taves et al. 2015, Hamden et al. 2019), we do not report local corticosterone elevation in the lymphoid organs at baseline compared to blood at PND5. Here, baseline animals were euthanized without anesthesia, and in previous studies, animals were briefly (<3 min) anesthetized with isoflurane before euthanasia (Taves et al. 2015, Hamden et al. 2019). It is well accepted that systemic GC levels in the blood require at least 3 min from the onset of a stressor to show significant increases above baseline levels (Romero & Reed 2005), but systemic ACTH levels increase more quickly. If ACTH stimulates local GC production, then it is possible that local GC levels rise before 3 min. This is an important methodological consideration for future studies of local GC levels.

The transition to similar stress-induced blood and local steroid levels at PND9, and to blood levels exceeding local levels at PND13, is consistent with increased responsiveness of the adrenals. As the adrenals increase in responsiveness, tissues have less need to locally produce corticosterone, as it can be more easily derived from the blood. Further, the greatest increases in lymphoid GC levels were observed at PND5, whereas greater increases in blood GC levels were observed at PND9 and PND13. Together, these data indicate that lymphoid production of corticosterone is greatest when adrenal production of corticosterone is lowest.

Multiple regression analysis using blood DOC, corticosterone, DHC and local DOC and DHC as predictors of local corticosterone levels demonstrates that the factors predicting local corticosterone levels change with age and stress. The strong ability of the model to predict local corticosterone levels at PND1 regardless of treatment, combined with the similar steroid levels across treatments, suggests that the same factors modulate both systemic and local GC levels at PND1. Our ability to predict baseline and oxygen-induced local corticosterone levels at PND5, 9, and 13 more frequently than corticosterone levels after isoflurane suggests that another factor is unaccounted for, such as tissue 11β-HSD1 activity. By quantifying enzyme activity within lymphoid organs, future studies can clarify whether local corticosterone levels are more influenced by synthesis from precursors or regeneration from metabolites.

The SHRP, local glucocorticoid production, and perinatal programming

During the SHRP, some stressors, such as maternal separation and endotoxin, produce slightly larger increases in blood corticosterone levels and have long-lasting effects on the immune system (Shanks et al. 2000, Hodgson et al. 2001, Bilbo et al. 2005, Walker et al. 2010). Other stressors, such as novelty and ether anesthesia, produce small increases in blood corticosterone levels (Tang & Phillips 1977, D’Amato et al. 1992). The present data raise the exciting possibility that stressors that only result in small increases to circulating corticosterone levels may also alter immunity via local production of corticosterone within lymphoid organs. Interestingly, neonatal stressors are commonly administered to mice at or near PND5, when the greatest increase in lymphoid corticosterone was observed in this study and when T and B cells are rapidly maturing (Bonneville et al. 2010, Mittelstadt et al. 2012, 2018). Increases in local corticosterone levels resulting from various stressors, regardless of whether corticosterone is adrenally or locally produced, can alter the critical process of lymphocyte selection (Mittelstadt et al. 2018). Higher GC levels during lymphocyte development lead to the selection of a more reactive T-cell repertoire, and thus, early-life stressors and local production may prepare individuals for harsh conditions later in life (Taves & Ashwell 2021).

The SHRP was characterized many decades ago (Sapolsky & Meaney 1986), but its purpose is still not well understood. Emerging evidence of local GC production during the SHRP suggests that the SHRP facilitates independent organ development. During neonatal development, some brain regions are highly sensitive to GC-induced apoptosis, and GC treatment during early development alters specific regions such as the hippocampus (Franks et al. 2020). In contrast, lymphoid organs require GCs to modulate development of a fully competent T-cell repertoire (Mittelstadt et al. 2018). The very low blood GC levels during the SHRP allow organs that require GCs to locally increase levels as needed, while allowing organs where high GC levels are harmful to maintain low levels. To increase our understanding of the purpose of the SHRP, future studies should examine other organ systems and other stressors, both psychological (e.g. isoflurane) and physiological (e.g. endotoxin).

Conclusions

GCs are produced within lymphoid organs, and local production may be particularly important during the SHRP because of reduced adrenal activity and very low blood corticosterone levels. The present data advance our knowledge of GC physiology in several key ways. First, we demonstrate that the HPA axis develops on a gradient of increasing adrenal responsivity, with low baseline and stress-induced systemic corticosterone levels at PND5. Second, local GC levels do not reflect blood GC levels during the SHRP. Local GC levels may be higher, similar, or lower depending on age and can increase more in organs than in blood. Third, local GC elevation is likely due to the local production of corticosterone. Together, these data provide important insights into the function of the SHRP and suggest a mechanism by which non-immunological stressors can modulate immune system development and have long-lasting implications for immunocyte function.

Supplementary materials

This is linked to the online version of the paper at https://doi.org/10.1530/JOE-21-0154.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants (RGPIN-2014-04884 and RGPIN-2019-04837) and Discovery Accelerator Supplement (RGDAS-2019-00033) and a Canada Foundation for Innovation Grant (32631) to K K S, a Four-Year Doctoral Fellowship from the University of British Columbia to J E H, NSERC CGS-M and CGS-D fellowships to M S and a NSERC URSA fellowship to K M G.

Acknowledgements

The authors thank Dr Matthew Taves and Dr Tamara Bodnar for critical editing of the manuscript, Dr Chunqi Ma for assistance with data acquisition, the Centre for Disease Modeling staff for animal husbandry, and the UBC Genotyping Facility.

References

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    Figure 1

    Simplified glucocorticoid synthetic pathway. Boxes indicate steroids measured in this study.

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    Figure 2

    In post-natal day 1 (PND1) mice, concentrations of (A) progesterone, (B) 11-deoxycorticosterone (DOC), (C) corticosterone, and (D) 11-dehydrocorticosterone (DHC) in whole blood, bone marrow, thymus, and spleen. Data are shown as mean ± s.e.m. There was no significant main effect of treatment or a tissue × treatment interaction, and thus, nopost hoc test was performed. n = 10 for all steroids and tissues. A full color version of this figure is available at https://doi.org/10.1530/JOE-21-0154.

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    Figure 3

    In post-natal day 5 (PND5) mice, concentrations of (A) progesterone, (B) 11-deoxycorticosterone (DOC), (C) corticosterone, and (D) 11-dehydrocorticosterone (DHC) in whole blood, bone marrow, thymus, and spleen. Data are shown as mean ± s.e.m. Tukey’s post hoc test was used to determine differences in baseline, oxygen, and isoflurane groups within each tissue, significant differences are denoted by letters. n = 10 for all steroids and tissues. For exact P values, see Supplementary Table 1. A full color version of this figure is available at https://doi.org/10.1530/JOE-21-0154.

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    Figure 4

    In post-natal day 9 (PND9) mice, concentrations of (A) progesterone, (B) 11-deoxycorticosterone (DOC), (C) corticosterone, and (D) 11-dehydrocorticosterone (DHC) in whole blood, bone marrow, thymus, and spleen. Data are shown as mean ± s.e.m. Tukey’s post hoc test was used to determine differences in baseline, oxygen, and isoflurane groups within each tissue, significant differences are denoted by letters. n = 10 for all steroids and tissues. For exact P values, see Supplementary Table 1. A full color version of this figure is available at https://doi.org/10.1530/JOE-21-0154.

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    Figure 5

    In post-natal day 13 (PND13) mice, concentrations of (A) progesterone, (B) 11-deoxycorticosterone (DOC), (C) corticosterone, (D) 11-dehydrocorticosterone (DHC) in whole blood, bone marrow, thymus, spleen. Data are shown as mean ± s.e.m.. Tukey’s post hoc test was used to determine differences in baseline, oxygen, and isoflurane groups within each tissue, significant differences are denoted by letters. n = 10 for all steroids and tissues. For exact P values, see Supplementary Table 1. A full color version of this figure is available at https://doi.org/10.1530/JOE-21-0154.

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    Figure 6

    In post-natal day (PND) 5, 9, and 13 mice, difference in tissue – blood corticosterone levels in (A) bone marrow, (B) thymus, (C) spleen. Data are show as mean ± s.e.m.. Tukey’s post hoc test was used to determine differences between PND5, PND9, and PND13 corticosterone levels within each treatment group, significant differences are denoted by letters. n = 10 for all ages and tissues. For exact P values, see Supplementary Table 4. A full color version of this figure is available at https://doi.org/10.1530/JOE-21-0154.

  • Avitsur R & Sheridan JF 2009 Neonatal stress modulates sickness behavior. Brain, Behavior, and Immunity 23 977985. (https://doi.org/10.1016/j.bbi.2009.05.056)

    • Search Google Scholar
    • Export Citation
  • Bilbo SD, Biedenkapp JC, Der-Avakian A, Watkins LR, Rudy JW & Maier SF 2005 Neonatal infection-induced memory impairment after lipopolysaccharide in adulthood is prevented via caspase-1 inhibition. Journal of Neuroscience 25 80008009. (https://doi.org/10.1523/JNEUROSCI.1748-05.2005)

    • Search Google Scholar
    • Export Citation
  • Boksa P 1997 Early developmental profiles of plasma corticosterone are altered by birth condition in the rat: a comparison of vaginal birth, cesarean section, and cesarean section with added anoxia. Pediatric Research 41 3443. (https://doi.org/10.1203/00006450-199701000-00006)

    • Search Google Scholar
    • Export Citation
  • Bonneville M, O’Brien RL & Born WK 2010 γδ T cell effector functions: a blend of innate programming and acquired plasticity. Nature Reviews: Immunology 10 467478. (https://doi.org/10.1038/nri2781)

    • Search Google Scholar
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  • Butte JC, Kakihana R & Noble EP 1972 Rat and mouse brain corticosterone. Endocrinology 90 10911100. (https://doi.org/10.1210/endo-90-4-1091)

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