TY - JOUR
AU1 - Russell, Ashley L
AU2 - Richardson, M Riley
AU3 - Bauman, Bradly M
AU4 - Hernandez, Ian M
AU5 - Saperstein, Samantha
AU6 - Handa, Robert J
AU7 - Wu, T John
AB - Abstract Traumatic brain injury (TBI) affects 10 million people worldwide, annually. TBI is linked to increased risk of psychiatric disorders. TBI, induced by explosive devices, has a unique phenotype. Over one-third of people exposed to blast-induced TBI (bTBI) have prolonged neuroendocrine deficits, shown by anterior pituitary dysfunction. Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is linked to increased risk for psychiatric disorders. Not only is there limited information on how the HPA axis responds to mild bTBI (mbTBI), sex differences are understudied. We examined central and peripheral HPA axis reactivity, 7 to 10 days after mbTBI in male and female mice. Males exposed to mbTBI had increased restraint-induced serum corticosterone (CORT), but attenuated restraint-induced corticotropin-releasing factor (CRF)/c-Fos-immunoreactivity (ir) in the paraventricular nucleus of the hypothalamus (PVN). Females displayed an opposite response, with attenuated restraint-induced CORT and enhanced restraint-induced PVN CRF/c-Fos-ir. We examined potential mechanisms underlying this dysregulation and found that mbTBI did not affect pituitary (pro-opiomelanocortin and CRF receptor subtype 1) or adrenal (11β-hydroxylase, 11β-dehydrogenase 1, and melanocortin 2 receptor) gene expression. mbTBI did not alter mineralocorticoid or glucocorticoid gene expression in the PVN or relevant limbic structures. In females, but not males, mbTBI decreased c-Fos-ir in non-neuroendocrine (presumably preautonomic) CRF neurons in the PVN. Whereas we demonstrated a sex-dependent link to stress dysregulation of preautonomic neurons in females, we hypothesize that mbTBI may disrupt limbic pathways involved in HPA axis coordination in males. Overall, mbTBI altered the HPA axis in a sex-dependent manner, highlighting the importance of developing therapies to target individual strategies that males and females use to cope with mbTBI. Blast-induced traumatic brain injuries (bTBIs), such as those from improvised explosive devices, account for ∼80% of traumatic brain injuries (TBIs) among service members (1). Mild TBI (mTBI) accounts for ∼75% of sustained injuries (2). mTBI is defined as a loss of consciousness (<30 minutes), posttraumatic amnesia (<24 hours), and minimal deficits in motor, verbal, and eye responses (3). Symptoms of mTBI may go unnoticed but result in long-term behavioral, cognitive, and emotional impairments. There is an increased prevalence of psychiatric disorders after mild bTBI (mbTBI) (4). Moreover, hypothalamic-pituitary-adrenal (HPA) axis hormonal dysregulation is one of the primary symptoms of neuropsychiatric disorders, such as anxiety, depression, and posttraumatic stress disorder (PTSD) (5, 6). TBI-induced dysregulation of the HPA axis has been observed in both clinical and preclinical studies (7–9). Therefore, HPA axis dysregulation may increase the risk for stress-related disorders after mbTBI. Physical and psychological stressors activate corticotropin-releasing factor (CRF) neurons in the paraventricular nucleus of the hypothalamus (PVN) (10, 11). CRF is released into the hypothalamo-hypophyseal portal vasculature to activate anterior pituitary corticotrophs and stimulate the release of ACTH. ACTH subsequently elicits the release of glucocorticoids [corticosterone (CORT); cortisol in humans, CORT in rats and mice] from the adrenal cortex (12). CORT regulates the HPA axis through the activation of neuronal mineralocorticoid (MR) and glucocorticoid (GR) receptors in a number of brain areas. These receptors are widely distributed throughout the brain, including the following limbic structures that are involved in HPA axis feedback: prelimbic (PrL) and infralimbic (IL) cortices (prefrontal cortex), bed nucleus of the stria terminalis (BNST), amygdala, and hippocampus (13–16). Proper coordination of the HPA axis is required for stress adaptation. In rodents, TBI is thought to cause acute and long-term HPA axis dysregulation, although the literature is somewhat inconsistent. For example, TBI has been shown to increase plasma ACTH and CORT acutely (17, 18). Different reports suggest that TBI can increase or decrease restraint-induced CORT chronically (7 or more days). Mild, controlled cortical impact injury attenuated CORT, 7 days postinjury, but heightened restraint-induced CORT, 34 and 70 days post-TBI (19). Fluid percussion injury decreased CORT, 54 days post-TBI (9). This inconsistency may be a result of differences in injury mechanism, severity, and time of recovery. Women have an increased prevalence of anxiety disorders and an increased diagnosis of psychiatric disorders after TBI (20, 21). In rodents, the inherent sex differences of the HPA axis are well established. Females have enhanced resting and stress-induced HPA axis activation (22–25). However, there is limited information on the sex-dependent differences in response to mbTBI. Central and peripheral HPA axis reactivity was examined, 7 to 10 days postinjury. Previous work has shown behavioral alterations and injury cascades at this 7- to 10-day period of recovery. For example, at this time point, there is evidence of increased cell death, astrogliosis, cerebral blood flow, axonal loss, as well as microglial infiltration and activation (26, 27). Additionally, deficits in motor, memory, acquisition of contextual fear, and anxiety are observed (19, 26, 28, 29). These behavioral disorders may be a result of a dysregulation of the HPA axis, which previous literature has shown to be present in various experimental TBI models during the subacute recovery period (19, 30, 31). Therefore, to address the sex difference in the HPA axis response to injury, adult male and female mice were exposed to mbTBI. Serum CORT, stress-relevant gene expression, and CRF neuronal activation, using c-Fos as a marker, were determined. Our data showed that the HPA axis of males and females responds differently to mbTBI, suggesting the use of different compensatory strategies. Materials and Methods Animals C57BL/6J Adult male (n = 60) and randomly cycling female (n = 72) C57BL/6J mice were purchased at 7 to 9 weeks of age from The Jackson Laboratory (Bar Harbor, ME; RRID: IMSR_JAX:000664; stock no. 000664). Upon arrival, C57BL/6J mice were acclimated to the facility for at least 1 week before experiments. CRF:tdTomato Male (n = 72) and randomly cycling females (n = 80) CRF:tdTomato mice were generated by crossing B6(Cg)-Crhtm(cre)Zjh/J (CRF-IRES-Cre; RRID: IMSR_JAX:012704; stock no. 012704; The Jackson Laboratory) mice and B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (Ai14; RRID: IMSR_JAX:007914; stock no. 007914; The Jackson Laboratory) mice. CRF:tdTomato mice are from the C57BL/6J background (32–34). Housing conditions Housing conditions were maintained at 22°C to 25°C, 50% humidity, on a 12-hour light:12-hour dark cycle (lights on at 0100 hours). Animals were provided ad libitum access to food and water. Animals were same-sex housed, two to three per cage. All handling and care of animals were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Procedures were approved by the Institutional Animal Care and Use Committee at the Uniformed Services University of the Health Sciences (Bethesda, MD). Experimental design There were four experiments in this study. For all experiments, HPA axis reactivity was assessed, 7 to 10 days postinjury. Unless otherwise stated, experiments were conducted between 0700 and 1100 hours (lights off at 1300 hours) to examine reactivity of the HPA axis when the HPA axis is at its nadir. Figure 1 depicts the experimental timeline for all of the following experiments. Figure 1. View largeDownload slide Experimental timeline. (A) Experimental timeline for C57BL/6J mice in experiments A and B. Adult male and female mice were given ∼7 days to acclimate before mbTBI. After a 7- to 10-day recovery period, animals in experiment A were exposed to a 20-minute restraint. Ten minutes following the conclusion of restraint, samples were collected for serum CORT and mRNA expression. In experiment B, C57BL/6J mice were exposed to the dexamethasone suppression test (DST) at the end of the lights-on phase (between 1230 and 1300 hours), and serum was collected for CORT analysis. (B) Experimental timeline for adult CRF:tdTomato mice in experiments C and D. In experiment C, mice were exposed to a 20-minute restraint, 7 to 10 days after the mbTBI. Samples were collected 45 minutes after the end of restraint to allow for peak c-Fos protein expression. The same experimental parameters applied for experiment D, except in this condition, animals received intraperitoneal injections of Fluoro-Gold (FG), 4 to 6 days before mbTBI. Exp, experiment. Figure 1. View largeDownload slide Experimental timeline. (A) Experimental timeline for C57BL/6J mice in experiments A and B. Adult male and female mice were given ∼7 days to acclimate before mbTBI. After a 7- to 10-day recovery period, animals in experiment A were exposed to a 20-minute restraint. Ten minutes following the conclusion of restraint, samples were collected for serum CORT and mRNA expression. In experiment B, C57BL/6J mice were exposed to the dexamethasone suppression test (DST) at the end of the lights-on phase (between 1230 and 1300 hours), and serum was collected for CORT analysis. (B) Experimental timeline for adult CRF:tdTomato mice in experiments C and D. In experiment C, mice were exposed to a 20-minute restraint, 7 to 10 days after the mbTBI. Samples were collected 45 minutes after the end of restraint to allow for peak c-Fos protein expression. The same experimental parameters applied for experiment D, except in this condition, animals received intraperitoneal injections of Fluoro-Gold (FG), 4 to 6 days before mbTBI. Exp, experiment. Experiment A C57BL/6J mice were randomly assigned to one of four treatment groups. The effect of injury (sham vs mbTBI) and restraint (no restraint vs restraint) on serum CORT was shown; mRNA expression was examined in males and females. Experiment B This experiment was designed as a 2 × 2 factorial design to examine the effect of mbTBI (sham vs mbTBI) on CORT secretion after the dexamethasone (DEX) suppression test [vehicle (VEH) vs DEX] in male and female C57BL/6J mice. Testing was conducted between 1230 and 1300 hours (approximately when lights came on) to examine the integrity of the physiological, nonmanipulated, and diurnal CORT rise. Experiment C CRF:tdTomato mice were randomly assigned to one of four experimental groups. The goal of experiment C was to examine the effect of injury (sham vs mbTBI) and restraint (no restraint vs restraint) on total CRF neuron activation in the PVN. Neuron activation was determined by c-Fos-immunoreactivity (ir) induction in male and female mice. Experiment D CRF:tdTomato mice were assigned to one of two experimental groups. In this experiment, 4 to 6 days before mbTBI, all animals received intraperitoneal injections of Fluoro-Gold (FG). Additionally, 7 to 10 days after mbTBI, all animals were exposed to restraint to activate the HPA axis. The goal of experiment D was to examine the effect of injury (sham vs mbTBI) on the percentage of activated (c-Fos+) CRF that were either endocrine projecting (FG+) or presumably preautonomic projecting (FG−). mbTBI Mice were exposed to mbTBI using the Advanced Blast Simulator (ABS; ORA Inc., Fredericksburg, VA; Fig. 2). The ABS consisted of three chambers: the driver chamber, the transition section, and the test chamber. The driver chamber was sealed with three acetate sheets (0.010 inches thick, 18 × 30 inches; Industrial Division Sales, Grafix, Inc., Maple Heights, OH) and two pet screen meshes (14 × 10 grids per square inch, wire diameter 0.025 inches; New York Wire). Increased pressure (by compressed air) in the driver chamber ruptured the acetate/mesh seal, allowing the blast wave to travel via the transition section to the test chamber (Fig. 2A and 2B). A pencil probe pressure gauge (model no. 137B23A; PCB Piezotronics, Depew, NY) recorded pressure at the level of the animal located in the test chamber. The mean pressure of the blast wave was not different between males (15.76 ± 0.09 psi) and females (15.74 ± 0.10 psi; Table 1 and Fig. 2C). Figure 2. View largeDownload slide (A) Schematic depiction of the ABS, viewed from the top and side (figure not drawn to scale). (A and B) The ABS consists of (A) a high-pressure driver (A′) membrane, (B) a transition section, (C) a test section, and (D) an end wave eliminator/muffler. (B) Montage of photographs of the ABS. (C) Representative profile of a blast overpressurization wave from the current study (courtesy of Dr. Yeonho Kim, Center for Neurosciencee and Regenerative Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD). Figure 2. View largeDownload slide (A) Schematic depiction of the ABS, viewed from the top and side (figure not drawn to scale). (A and B) The ABS consists of (A) a high-pressure driver (A′) membrane, (B) a transition section, (C) a test section, and (D) an end wave eliminator/muffler. (B) Montage of photographs of the ABS. (C) Representative profile of a blast overpressurization wave from the current study (courtesy of Dr. Yeonho Kim, Center for Neurosciencee and Regenerative Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD). Table 1. Parameters of Sham and Injury Procedures in Male and Female Mice Condition Anesthesia Length, s Blast Pressure, psi Male sham 340.23 ± 7.28 N/A Male blast 345.98 ± 7.18 15.76 ± 0.09 Female sham 325.20 ± 6.17 N/A Female blast 348.15 ± 7.76a 15.74 ± 0.10 Condition Anesthesia Length, s Blast Pressure, psi Male sham 340.23 ± 7.28 N/A Male blast 345.98 ± 7.18 15.76 ± 0.09 Female sham 325.20 ± 6.17 N/A Female blast 348.15 ± 7.76a 15.74 ± 0.10 Table presents length of anesthetic (s) and the pressure of the blast wave (psi), where applicable. Data represent means ± SEM. a P < 0.05 comparing sham vs blast in respective sex. View Large Table 1. Parameters of Sham and Injury Procedures in Male and Female Mice Condition Anesthesia Length, s Blast Pressure, psi Male sham 340.23 ± 7.28 N/A Male blast 345.98 ± 7.18 15.76 ± 0.09 Female sham 325.20 ± 6.17 N/A Female blast 348.15 ± 7.76a 15.74 ± 0.10 Condition Anesthesia Length, s Blast Pressure, psi Male sham 340.23 ± 7.28 N/A Male blast 345.98 ± 7.18 15.76 ± 0.09 Female sham 325.20 ± 6.17 N/A Female blast 348.15 ± 7.76a 15.74 ± 0.10 Table presents length of anesthetic (s) and the pressure of the blast wave (psi), where applicable. Data represent means ± SEM. a P < 0.05 comparing sham vs blast in respective sex. View Large Mice were anesthetized by isoflurane inhalation (isoflurane, USP; Baxter, Deerfield, IL), secured and mounted prone in the test chamber. Sham animals were anesthetized but were not exposed to the blast injury. There were no differences in time of anesthesia exposure between males and females. Anesthetic exposure time was less in sham females than mbTBI females (Table 1). Immediately after sham or injury procedures, mice were observed for righting reflex and returned to the home cage for recovery. Righting reflex Righting reflex was measured on the day of injury. After removal from anesthesia, the animals were placed on their backs in the home cage. The time it took for the animal to return to an upright position (forepaws touching the ground) was recorded as the time to right (35). DEX suppression test C57BL/6J male and female mice were injected subcutaneously, 7 to 10 days after mbTBI with DEX (Sigma, St. Louis, MO), at a dose of 30 μg/kg at 0600 hours, following previously established protocols (36). Animals were injected between 1230 and 1300 hours (lights off at 1300). Retrograde FG injection Male and female CRF:tdTomato mice were intraperitoneally injected with FG (Fluorochrome, LLC, Denver, CO), dissolved in 0.9% saline at a dose of 15 mg/kg (37). Neurons that project outside of the blood brain barrier will readily take up FG. As a result of their axon terminations, neuroendocrine-projecting CRF populations are FG+ (32). Animals were injected 4 to 6 days before mbTBI. All animals that received FG injections were exposed to restraint, 7 to 10 days after mbTBI. Restraint Animals were individually restrained for 20 minutes in clear Plexiglas tubes (3.81 × 10.16 cm; Stoelting Company, Wood Dale, IL), 7 to 10 days after sham or mbTBI procedures. Mice were anesthetized for rapid decapitation (C57BL/6J) or intracardiac perfusion (CRF:tdTomato) at 10 minutes or 45 to 60 minutes after the conclusion of restraint, respectively. Tissue collection and processing For experiments A and B, C57BL/6J mice were deeply anesthetized with carbon dioxide inhalation and subsequently euthanized by decapitation. Pituitary, adrenals, liver, and brain were collected and immediately frozen. Blood was collected and immediately placed on ice and centrifuged, and serum was harvested. Brains were removed and flash frozen in 2-methylbutane (−40°C). All were stored at −80°C until analyzed. Brains were sectioned at 250 μm (HM525 Crytostat; Thermo Fisher Scientific, Waltham, MA). Either 1 mm (Integra York PA, Inc., York, PA)- or 0.5 mm (Harris Uni-Core; Sigma, St. Louis, MO)-diameter tissue punches were taken to isolate specific regions. The following regions were collected (respective bregma coordinates based on the Franklin and Paxinos Mouse Brain Atlas): PrL (1.98 to 1.34 mm), IL (1.98 to 1.34 mm), anterior BNST (aBNST; 0.62 to 0.02 mm), posterior BNST (pBNST; −0.10 to −0.58 mm), PVN (−0.22 to −1.22 mm), amygdala (−0.70 to −2.30 mm), dorsal hippocampus (−1.34 to −2.80 mm), and ventral hippocampus (−2.70 to −3.80 mm). For experiments C and D, CRF:tdTomato mice were deeply anesthetized with carbon dioxide inhalation and sequentially intracardially perfused with 0.9% saline, followed by 100 mL of 4% paraformaldehyde (PFA)-buffered by a 0.1-M phosphate solution (PFA; pH 7.4). Brains were collected and postfixed in PFA overnight. Subsequently, brains were incubated in an increasing graded, buffered sucrose gradient (12%, 15%, and 18% sucrose in 0.1 M phosphate buffered saline, respectively). Brains were sectioned at 40 μm (HM525 Cryostat; Thermo Fisher Scientific, Waltham, MA). Sections were stored in 0.1 M PBS containing 0.05% sodium azide (Sigma) until use. CORT assay Serum CORT was measured using a commercially available ELISA (catalog no. K014; Arbor Assays, Ann Arbor, MI), following the manufacturer’s instructions. Samples were diluted to a final concentration of 1:150 and analyzed in duplicate. Subsequent to dilution, samples were treated with the dissociation buffer and heated to 60°C. Changes in binding were measured using a plate reader (Victor V3; PerkinElmer, Waltham, MA), which read absorbance at a wavelength of 450 nm. Values were obtained by comparison, with a standard curve ranging from 78.125 to 10,000 pg/mL. Intra-assay coefficient of variance was 7.5%, and the minimum detectable CORT concentration was 16.9 pg/mL. Gene expression RNA extraction Total RNA was extracted from pituitary, adrenals, liver, and brain regions of C57BL/6J mice, using a commercially available RNA prep kit (Direct-zol RNA MiniPrep Kit; catalog no. 11-331; Zymo Research, Tustin, CA), following the manufacturer’s protocol. In brief, the tissue was homogenized in 1 mL (pituitary, adrenal, liver) or 500 μL (brain) Ribozol (Thermo Fisher Scientific) before column extraction. The total RNA concentration was determined by a spectrophotometer (NanoDrop Lite; Thermo Fisher Scientific), measuring absorbance at 260 and 280 nm, and the purity was determined from the 260:280 ratio. cDNA synthesis cDNA was synthesized from purified RNA using reverse transcription (Thermo Scientific Maxima Reverse transcription kit; catalog no. K1671; Thermo Fisher Scientific). Real-time quantitative PCR Pituitary, adrenals, and liver mRNA expression were measured by quantitative PCR, following previously established protocols (38). In brief, 2 ng cDNA template was amplified using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA). Primer sequences for pituitary [pro-opiomelanocortin (POMC) and CRF receptor subtype 1 (CRFR1)], adrenal [11β-hydroxylase (11βOHase), 11β-hydroxysteroid dehydrogenase 1 (11βHSD1), and melanocortin 2 receptor (Mc2r)], and liver [corticosteroid-binding globulin (CBG)] were designed to have a common annealing temperature of 60°C using the National Center for Biotechnology Information Primer blast (Supplemental Table 1) (39). Each run consisted of an initial denaturation (3 minutes at 95°C), followed by 40 cycles that included denaturation (10 seconds at 95°C), annealing (30 seconds at 60°C), and extension (30 seconds at 72°C) using the CFX Connect Real-Time System (Bio-Rad). Melt-curve analysis was performed after each run to ensure a single amplicon. All data were normalized to TATA box-binding protein and expressed relative to the control group. The 2−ΔΔCT method was used for analysis. All samples were analyzed in duplicate. Droplet digital PCR The absolute copy number of MR and GR was quantified using droplet digital PCR in the following brain regions: PrL, IL, PVN, aBNST, pBNST, amygdala, and dorsal/ventral hippocampus. Primer sequences were generated from National Center for Biotechnology Information Primer blast (Supplemental Table 1) (39). Each 20-μL droplet digital PCR reaction contained EvaGreen Supermix (1× solution; Bio-Rad), forward and reverse primers (200 nM each), and cDNA template (2 ng). Droplets were generated using the Automated Droplet Generator (Bio-Rad). Thermal cycling conditions included the following: enzyme activation (5 minutes at 95°C), followed by 40 cycles of denaturation (30 seconds at 95°C), annealing/extension (1 minute at 60°C), and one cycle of signal stabilization (5 minutes at 4°C, followed by 5 minutes at 90°C). Droplets were read using the QX200 Droplet Reader and QuantaSoft software (Bio-Rad). The number of copies per nanogram cDNA template was used for analysis. Immunohistochemistry c-Fos Sections (40 μm) were processed for c-Fos-ir following previously established protocols (40). Sections were washed in 0.1 M PBS containing 0.5% Triton X-100 (PBS-T). The tissue was then blocked in PBS containing 5% normal goat serum, 1% BSA, for 1 hour at room temperature. Sections were incubated in anti-c-Fos antibody (1:1500; RRID: AB_2631318; EMD Millipore, Billerica, MA), overnight at 4°C, with gentle agitation. The tissue was washed with PBS-T and incubated with goat anti-rabbit IgG coupled to Alexa Fluor 488 antibody (1:1000; RRID: AB_2338052; Jackson ImmunoResearch Laboratories, West Grove, PA) for 2 hours at room temperature. The tissue was washed, mounted, and coverslipped with Prolong Gold anti-fade reagent (Invitrogen, Eugene, OR). FG and c-Fos Sections were washed and blocked following the above procedure. Sections were incubated in anti-FG (1:50,000; RRID: AB_2314408; Fluorochrome, LLC, Denver, CO) and diluted in company-recommended diluent (50 nM potassium PBS, 0.4% Triton, 1% BSA, and 1% normal goat serum) overnight at 4°C. The tissue was washed with PBS-T and incubated with an Alexa Fluor 647 AffiniPure F(ab′)2 Fragment Goat Anti-Mouse IgG (1:700; RRID: AB_2338058; Jackson ImmunoResearch Laboratories) for 2 hours at room temperature. Sections were then placed in anti-c-Fos (1:1500) following the above protocol. For labeling determination, counts were conducted on nuclear c-Fos and cytoplasmic FG labeling. Antibody information is found in Table 2. Table 2. Antibody Information Target Specificity Source, Catalog No. Species Raised in; Polyclonal or Monoclonal Working Dilution c-Fos N-terminus EMD Millipore, ABE457 Rabbit; polyclonal 1:1500 FG Fluorochrome Rabbit; polyclonal 1:50,000 Target Specificity Source, Catalog No. Species Raised in; Polyclonal or Monoclonal Working Dilution c-Fos N-terminus EMD Millipore, ABE457 Rabbit; polyclonal 1:1500 FG Fluorochrome Rabbit; polyclonal 1:50,000 View Large Table 2. Antibody Information Target Specificity Source, Catalog No. Species Raised in; Polyclonal or Monoclonal Working Dilution c-Fos N-terminus EMD Millipore, ABE457 Rabbit; polyclonal 1:1500 FG Fluorochrome Rabbit; polyclonal 1:50,000 Target Specificity Source, Catalog No. Species Raised in; Polyclonal or Monoclonal Working Dilution c-Fos N-terminus EMD Millipore, ABE457 Rabbit; polyclonal 1:1500 FG Fluorochrome Rabbit; polyclonal 1:50,000 View Large Microscopy All images were acquired with a Zeiss 700 Confocal Laser-Scanning Microscope using a 20× objective (Zeiss, Thornwood, NY). The number of CRF-positive, c-Fos-positive, and where applicable, FG-positive cells was manually counted from z-stack images compiled using Zeiss Zen 2 software (RRID: SCR_013672). For each animal, on average, six PVN sections at 40 μm were analyzed to obtain counts from every subdivision (anterior, medial, and posterior) of the PVN. For data analysis, all subdivisions were combined, and analysis was conducted on the total rostral-to-caudal span of the PVN. All images were coded and counted by two independent counters using ImageJ software (National Institutes of Health, Bethesda, MD; RRID: SCR_003070). Any count above a 15% difference between independent counters was recounted by a third counter, and values >15% difference were excluded. The data were compiled, and counts were averaged. Statistical analysis An initial three-way ANOVA (SPSS, Armonk, NY; injury × restraint × sex) showed that males and females differed in response to injury and restraint. Therefore, the primary analysis was conducted separately. Data were analyzed separately in males and females using two-way ANOVA (GraphPad Software, La Jolla, CA). Fisher least significant difference test was used for post hoc analysis when a significant effect was present. P < 0.05 was considered significant. The DEX suppression test (conducted between 1230 and 1300 hours) and FG retrograde labeling (conducted in restraint animals) were used to examine the effect of mbTBI only. These data sets were analyzed using unpaired t tests in male and female (GraphPad Software). P < 0.05 was considered significant. Results Effect of mbTBI on righting reflex Righting reflex was used to measure return to consciousness (Fig. 3A). Two-way ANOVA showed that males and females exposed to mbTBI had increased time to right compared with the respective sham group (P < 0.05). Figure 3. View largeDownload slide Effect of mbTBI on righting reflex and body weight gain. (A) Males and females exposed to mbTBI had an increased time to right compared with the respective sham group. (B) mbTBI had no effect on body weight gain (grams), 7 to 10 days postinjury, in males or females (n = 17 to 18 per treatment group). Days −8 to −2 represent preblast body weight. Days 2 to 4 represent postblast body weight. Body weight was also measured on the day of mbTBI (Day 0). Data represent means ± SEM; *P < 0.05. Figure 3. View largeDownload slide Effect of mbTBI on righting reflex and body weight gain. (A) Males and females exposed to mbTBI had an increased time to right compared with the respective sham group. (B) mbTBI had no effect on body weight gain (grams), 7 to 10 days postinjury, in males or females (n = 17 to 18 per treatment group). Days −8 to −2 represent preblast body weight. Days 2 to 4 represent postblast body weight. Body weight was also measured on the day of mbTBI (Day 0). Data represent means ± SEM; *P < 0.05. Effect of mbTBI on body weight change Body weight was used to assess health and wellbeing after injury (Fig. 3B). Animals were weighed every other day before (day −8 to −2) and after (day 2 to 6) injury. There was no difference in baseline weights. mbTBI did not alter body weight gain at any point during the 7- to 10-day recovery period in males or females (P > 0.10). Effect of mbTBI on serum CORT Response to restraint Serum CORT was assayed to measure baseline and restraint-induced HPA axis reactivity during the nadir of the CORT cycle (Fig. 4; experiment A). Two-way ANOVA showed that in males, there was a main effect of injury (P < 0.05), a main effect of restraint (P < 0.05), and no significant interaction. Post hoc analysis showed that restraint increased CORT, regardless of injury (P < 0.05). mbTBI had no effect on baseline CORT but enhanced restraint-induced CORT compared with sham males (P < 0.05; Fig. 4A). Figure 4. View largeDownload slide Effect of mbTBI on baseline and restraint-induced serum CORT. (A and B) Restraint increased CORT in males and females, regardless of injury. There was no effect of mbTBI on baseline CORT in males or females. (A) In males, mbTBI increased restraint-induced CORT. (B) In females, mbTBI attenuated restraint-induced CORT. Data represent means ± SEM; *P < 0.05. Figure 4. View largeDownload slide Effect of mbTBI on baseline and restraint-induced serum CORT. (A and B) Restraint increased CORT in males and females, regardless of injury. There was no effect of mbTBI on baseline CORT in males or females. (A) In males, mbTBI increased restraint-induced CORT. (B) In females, mbTBI attenuated restraint-induced CORT. Data represent means ± SEM; *P < 0.05. In females, a two-way ANOVA revealed a significant interaction of injury and restraint (P < 0.05) and a main effect of restraint (P < 0.05). Post hoc analysis showed that restraint increased CORT regardless of injury (P < 0.05). mbTBI did not alter baseline CORT but attenuated restraint-induced CORT compared with sham females (P < 0.05; Fig. 4B). DEX suppression test To test for peripheral HPA axis feedback, animals were administered a DEX suppression test, and CORT was measured at its diurnal peak (1230 to 1300 hours; Fig. 5; experiment B). In both males and females, there was a main effect of DEX treatment (P < 0.05). DEX administration attenuated CORT in males (P < 0.05) and females (P < 0.05) compared with respective VEH-treated animals. In males, mbTBI had no effect on VEH or DEX treatment (P > 0.10; Fig. 5A). In females, mbTBI had no effect on DEX suppression of CORT (P > 0.10). However, mbTBI increased the diurnal CORT peak in VEH-treated animals (P < 0.05; Fig. 5B). Overall, mbTBI did not affect CORT suppression to the DEX suppression test in males or females. Figure 5. View largeDownload slide Effect of mbTBI on the DEX suppression test. (A and B) DEX attenuated diurnal CORT regardless of injury. (A) mbTBI had no effect on the DEX suppression in males. (B) In females, mbTBI increased the diurnal CORT peak but had no effect on DEX suppression. Data represent means ± SEM; *P < 0.05. Figure 5. View largeDownload slide Effect of mbTBI on the DEX suppression test. (A and B) DEX attenuated diurnal CORT regardless of injury. (A) mbTBI had no effect on the DEX suppression in males. (B) In females, mbTBI increased the diurnal CORT peak but had no effect on DEX suppression. Data represent means ± SEM; *P < 0.05. Effect of mbTBI on gene expression in the peripheral HPA axis HPA-relevant gene expression was examined in the pituitary, adrenals, and liver (Supplemental Table 2). There was no effect of either mbTBI or restraint on pituitary (POMC, CRFR1) or adrenal (11β-OHase, 11β-HSD1, and Mc2r) genes in males or females (P > 0.10). A two-way ANOVA revealed a main effect of injury on liver CBG mRNA expression in males (P < 0.05). mbTBI increased restraint-induced CBG mRNA expression compared with sham animals (P < 0.05). Effect of mbTBI on MR and GR mRNA expression The response of MR and GR mRNA expression to mbTBI was analyzed in the limbic-HPA axis, 7 to 10 days postinjury. PVN A two-way ANOVA showed that there were no main effects of injury or restraint on male (Fig. 6A) or female (Fig. 6B) MR mRNA expression in the PVN (P > 0.10). Likewise, there were no main effects of injury or restraint on male (Fig. 6C) or female (Fig. 6D) GR mRNA expression in the PVN (P > 0.10). Figure 6. View largeDownload slide Effect of mbTBI on MR and GR mRNA expression in the PVN. mbTBI had no effect on MR mRNA expression in either (A) males or (B) females. Injury also had no effect on GR mRNA expression in (C) males or (D) females. Data represent means ± SEM. Figure 6. View largeDownload slide Effect of mbTBI on MR and GR mRNA expression in the PVN. mbTBI had no effect on MR mRNA expression in either (A) males or (B) females. Injury also had no effect on GR mRNA expression in (C) males or (D) females. Data represent means ± SEM. MR mRNA expression in limbic structures In males, there was no effect of injury or restraint on MR mRNA expression in the PrL, IL, aBNST, pBNST, amygdala, dorsal hippocampus, or ventral hippocampus (P > 0.10; Supplemental Table 3). Likewise, in females, neither injury nor restraint had an effect on MR mRNA expression in the aforementioned structures (P > 0.10; Supplemental Table 3). GR mRNA expression in limbic structures There was no effect of mbTBI or restraint on GR mRNA expression in any of the previously mentioned limbic structures in males or females (P > 0.10; Supplemental Table 4). MR/GR mRNA expression ratio in limbic structures The ratio of MR/GR mRNA expression was also analyzed to determine changes in relative expression (Supplemental Table 5). There was no effect of injury on the MR/GR ratio in the PrL, IL, aBNST, pBNST, amygdala, or ventral hippocampus in males or females (P > 0.10). In males, there was an effect of restraint in the dorsal hippocampus. In shams, restraint decreased the MR/GR ratio (P < 0.05). Effect of mbTBI on CRF activation in the PVN CRF neuron activation in the PVN was quantified by visualization of c-Fos-ir (Fig. 7). There was a main effect of restraint in males and females. In both sham and injured males and females, restraint increased CRF activation compared with the respective no-restraint group (P < 0.05). In males, mbTBI attenuated the restraint-induced increase in CRF activation (P < 0.05; Fig. 7C). In contrast, mbTBI increased restraint-induced CRF activation in females (P < 0.05; Fig. 7D). Figure 7. View largeDownload slide Effect of mbTBI on CRF activation in the PVN. Photomicrographs showing confocal images at (A) ×63 and (B) ×10 magnification. (A) Example of CRF (red) and c-Fos (green) colocalization. (Colocalized neurons are represented by yellow.) (B) Representative images of CRF and c-Fos in the PVN of males (upper) and females (lower). (C and D) Restraint increased CRF activation regardless of injury. (C) In males, mbTBI attenuated restraint-induced CRF activation. (D) In females, mbTBI increased restraint-induced CRF activation. Data represent means ± SEM; *P < 0.05. V, third ventricle. Figure 7. View largeDownload slide Effect of mbTBI on CRF activation in the PVN. Photomicrographs showing confocal images at (A) ×63 and (B) ×10 magnification. (A) Example of CRF (red) and c-Fos (green) colocalization. (Colocalized neurons are represented by yellow.) (B) Representative images of CRF and c-Fos in the PVN of males (upper) and females (lower). (C and D) Restraint increased CRF activation regardless of injury. (C) In males, mbTBI attenuated restraint-induced CRF activation. (D) In females, mbTBI increased restraint-induced CRF activation. Data represent means ± SEM; *P < 0.05. V, third ventricle. Effect of mbTBI on activation of FG+ and FG− CRF populations To determine activated CRF neurons (c-Fos+) in the PVN, animals received the retrograde tracer, FG, to label neuroendocrine populations (Fig. 8). All animals received restraint. In males, mbTBI did not alter the percentage of activated FG+ CRF neurons (P > 0.10; Fig. 8B) or activated FG− CRF neurons (P > 0.10; Fig. 8C). However, in females, we observed an effect of mbTBI on the percentage of activated CRF neurons. mbTBI decreased the percent of activated FG- CRF (non-neuroendocrine) populations (P < 0.05; Fig. 8C). There was also a nonsignificant trend to an increase of activated FG+ CRF neurons (P = 0.057; Fig. 8B). Figure 8. View largeDownload slide Effect of mbTBI on activated FG+ and FG− CRF populations. (A) Photomicrographs of confocal images at ×20 magnification throughout the rostral-to-caudal span of the PVN. Images represent colocalization patterns of CRF (red), c-Fos (green), and FG (blue) in the following subnuclei of the PVN: anterior parvocellular PVN (pPVN; PaAP), medial pPVN (PaMP), and posterior pPVN (PaPO). (B) mbTBI had no effect on activated FG+ or FG− CRF neurons in males. (C) In females, mbTBI decreased activated FG− CRF (preautonomic) neurons. Data represent means ± SEM; *P < 0.05. V, third ventricle. Figure 8. View largeDownload slide Effect of mbTBI on activated FG+ and FG− CRF populations. (A) Photomicrographs of confocal images at ×20 magnification throughout the rostral-to-caudal span of the PVN. Images represent colocalization patterns of CRF (red), c-Fos (green), and FG (blue) in the following subnuclei of the PVN: anterior parvocellular PVN (pPVN; PaAP), medial pPVN (PaMP), and posterior pPVN (PaPO). (B) mbTBI had no effect on activated FG+ or FG− CRF neurons in males. (C) In females, mbTBI decreased activated FG− CRF (preautonomic) neurons. Data represent means ± SEM; *P < 0.05. V, third ventricle. Discussion The results of the current study showed that mbTBI dysregulated the HPA axis in male and female mice. In males, mbTBI increased restraint-induced CORT but decreased restraint-induced CRF neuron activation. Conversely, in females, mbTBI decreased restraint-induced CORT but increased restraint-induced CRF neuron activation. The opposing trends of CORT and CRF activation indicated that mbTBI does indeed dysregulate the HPA axis. The experiments in this study sought to determine at what point in the circuitry this dysregulation may be occurring. Males and females had opposite CORT responses to restraint after mbTBI In males, mbTBI increased restraint-induced CORT levels but attenuated restraint-induced CORT release in females. To determine if the dysregulation occurred at the level of the peripheral HPA axis, we measured expression of genes involved in ACTH and CORT synthesis and secretion. Our present results showed no changes in pituitary (CRFR1, POMC) or adrenal (11β-OHase, 11β-HSD1, Mc2r) gene expression. Animals were administered the DEX suppression test after mbTBI to examine peripheral feedback. DEX suppressed the diurnal peak in CORT, suggesting intact feedback. Clinically, there is increased prevalence of acute and chronic hypopituitarism (7, 41) and hypoadrenalism (42–44). Whereas we did not measure ACTH release from the pituitary, gene-expression analysis allowed for an alternate route of investigation. With no apparent peripheral gene changes or alterations in the DEX suppression test, CORT dysregulation after mbTBI may be a result of a central disruption in feedback. mbTBI did not alter MR or GR mRNA expression mbTBI did not alter MR or GR mRNA expression in the PVN or stress-relevant limbic structures. HPA axis feedback is governed by CORT binding to MR and GR. Feedback regulation occurs at the level of the PVN (45) or via PVN projecting limbic structures. Our results are supported by previous studies, which revealed no change in GR protein levels after injury. The lack of MR/GR change suggested that the expression of limbic receptors involved in feedback regulation of the PVN are intact after mbTBI. mbTBI had opposing effects on CRF activation in males and females CRF colocalization with c-Fos-ir measured neuronal activation in the PVN. Acute restraint increased CRF neuron activation in the PVN of males and females, as previously shown (25, 46). mbTBI attenuated restraint-induced CRF neuron activation in males but enhanced CRF neuron activation in females. This discrepancy to CORT response may arise from a sex-dependent dysregulation at the level of CRF neurons in the PVN. The PVN regulates neuroendocrine, autonomic, and behavioral responses to stress (47, 48). Our anatomical understanding of the mouse PVN is extrapolated from rat studies (49). CRF neurons in the PVN are divided into two categories: parvocellular neuroendocrine neurons and non-neurosecretory (presumably, preautonomic) neurons (50–53). Anterior and medial subdivisions of the parvocellular PVN (pPVN) project to the median eminence (54, 55) to regulate endocrine response. Projections from the dorsomedial cap and ventral and posterior subdivisions of the rat pPVN are sent to sympathetic neurons of the intermediolateral nucleus of the spinal cord and presympathetic neurons of the rostroventrolateral medulla (51, 56–59), regulating autonomic function. We observed the highest density of activated CRF neurons in the anterior and medial subdivisions of the pPVN in both males and females (not quantified), suggesting the primary output of CRF neurons is neuroendocrine. However, it is important to examine the impact of mbTBI on the activation of neuroendocrine and non-neuroendocrine CRF populations to address projection integrity after injury. Peripheral FG injection labels hypophysiotrophic neurons that project outside of the blood brain barrier. Therefore, subsequent cytoplasmic labeling of FG distinguished neuroendocrine from non-neuroendocrine CRF populations. mbTBI decreased the percentage of activated non-neuroendocrine (presumably, preautonomic projecting CRF neurons in females but not males). This suggests a decreased use of the preautonomic system to cope with a stressor after mbTBI in females. Previous studies showed enhanced autonomic responses to acute stress in females (60–62). In our sham animals, females had increased restraint-induced activation of non-neuroendocrine (preautonomic) CRF neurons compared with males, paralleling evidence for an enhanced role of the autonomic nervous system. mbTBI reduced non-neuroendocrine (preautonomic) CRF activation in females, potentially disrupting this circuitry. Preautonomic PVN neurons are under constant inhibition by peri-PVN GABAergic interneurons (63). Therefore, integrity of the GABA circuitry is crucial for proper coordination of PVN outputs. GABA and its receptors are dysregulated after experimental TBI (64, 65). It is unclear if mbTBI mimics these effects in the same fashion as other forms of TBI. However, if mbTBI disrupted the inhibitory tone on the PVN, then this may interfere with sympathetic and presympathetic CRF projections to brainstem structures in females. In addition to the role in sympathetic reactivity, GABAergic projections to the PVN are pivotal for proper coordination of the HPA axis. Neuroendocrine-projecting CRF neurons are surrounded by a shell of GABAergic neurons involved in neuronal inhibition (63). Limbic structures project indirectly to the PVN via two primarily GABAergic regions: the BNST and the peri-PVN (13, 66). In males, disruption in limbic GABAergic projections may also manifest through HPA axis dysregulation. Further studies are needed to elucidate the neurotransmitter and neuropeptide involvement in the limbic projections to the HPA axis, as these might have sex differences that are not reflected by MR and GR levels. Where in females, we observed an autonomic connection, we hypothesize that mbTBI may induce a greater degree of functional dysregulation of limbic structures in males. This may ultimately disrupt the integrity of projections that normally mediate a physiological stress response to maintain homeostasis. Stress responsivity is a result of both organizational and activational roles of gonadal hormones (67), potentially explaining differences in stress sensitivity. In experiments not shown, we observed no differences between naturally cycling females in the proestrus phase (high estrogen) and diestrus phase (low estrogen) of the estrus cycle in CORT secretion or CRF activation (A.L. Russell, unpublished data, 2017). The current data suggest that there are inherent sex differences in the neural circuitry that are independent of steroid activation. Rather, there may be inherent organizational differences in male and female HPA axis reactivity, which may impact the stress response to mbTBI later in life. Females had an enhanced peak in diurnal CORT after mbTBI In females, mbTBI enhanced the diurnal CORT peak. Many TBI patients experience a sleep disturbance after injury (68). In a fluid percussion injury TBI model, the circadian timing of clock genes is dysregulated (69). Circadian disruptions, such as dysregulation of natural CORT rhythmicity, are linked to depression, anxiety, PTSD, and cardiovascular diseases (70–73). The inherent sex differences in suprachiasmatic nucleus morphology, gonadal steroid receptor distribution, electrical activity, and efferent projections (74, 75) may explain the sex-dependent disruptions of CORT rhythmicity observed after mbTBI. Different experimental TBI models may elicit different HPA axis responses Few studies have examined bTBI but rather, have used other animal models of TBI. Additionally, few studies have examined male-female differences. Dependent on the experimental TBI model, restraint can increase (30) or decrease (19) CORT throughout the chronic recovery period (7 or more days after injury). Twenty-one days after injury, forced swim stress attenuated CORT chronically (19). Our data conflict with previous literature potentially as a result of inherent differences in our blast-induced injury model. Different experimental TBI models may alter different neuroanatomical pathways. The mechanisms of the physical wave and the diffuse nature of the injury make mbTBI distinct from other forms of closed head injury (76). Among military populations, bTBI is a primary cause of trauma (1) and a “signature wound of the war” (77). PTSD, depression, and anxiety are observed in more than one-third of veterans (78, 79). As a result of the risk of developing psychiatric disorders after bTBI (1), it is important to understand the underlying physiological response to this form of injury. Conclusions In summary, present findings demonstrate that mbTBI altered the HPA axis and that this may stem from a disruption at the level of the central stress regulator: the PVN. The HPA axis is susceptible to TBI in both males and females; however, the profile of reactivity differed. We conclude that males and females use different strategies to cope with mbTBI-induced HPA axis dysregulation. Abbreviations: Abbreviations: 11β-HSD1 11β-hydroxysteroid dehydrogenase 1 11β-OHase 11β-hydroxylase aBNST anterior bed nucleus of the stria terminalis ABS Advanced Blast Simulator BNST bed nucleus of the stria terminalis bTBI blast-induced traumatic brain injury CBG corticosteroid-binding globulin CORT corticosterone CRF corticotropin-releasing factor CRFR1 corticotropin-releasing factor receptor subtype 1 DEX dexamethasone FG Fluoro-Gold GR glucocorticoid receptor HPA hypothalamic-pituitary-adrenal IL infralimbic ir immunoreactivity Mc2r melanocortin 2 receptor MR mineralocorticoid receptor mTBI mild traumatic brain injury mbTBI mild blast-induced traumatic brain injury pBNST posterior bed nucleus of the stria terminalis PBS-T PBS containing 0.5% Triton X-100 PFA paraformaldehyde POMC pro-opiomelanocortin pPVN parvocellular paraventricular nucleus of the hypothalamus PrL prelimbic PTSD posttraumatic stress disorder PVN paraventricular nucleus of the hypothalamus TBI traumatic brain injury VEH vehicle Acknowledgments We thank Dr. Amanda Fu, Dr. Yeonho Kim, Dr. Irwin Lucki, Dr. Cara Olsen, Sorana Raiciulescu, Dr. Aviva Symes, and Dr. Dennis McDaniel for their expert assistance. Financial Support: This work was funded by the Department of Defense in the Center for Neuroscience and Regenerative Medicine. Disclosure Summary: The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences. The authors have no conflicts of interest to disclose. References 1. Licona NE , Chung JS , Poole JH , Salerno RM , Laurenson NM , Harris OA . Prospective tracking and analysis of traumatic brain injury in veterans and military personnel . Arch Phys Med Rehabil . 2017 ; 98 ( 2 ): 391 – 394 . Google Scholar CrossRef Search ADS PubMed 2. National Center for Injury Prevention and Control . Report to Congress on Mild Traumatic Brain Injury in the United States: Steps to Prevent a Serious Public Health Problem . Atlanta, GA : Centers for Disease Control and Prevention ; 2003 . 3. 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TI - Differential Responses of the HPA Axis to Mild Blast Traumatic Brain Injury in Male and Female Mice
JF - Endocrinology
DO - 10.1210/en.2018-00203
DA - 2018-04-25
UR - https://www.deepdyve.com/lp/oxford-university-press/differential-responses-of-the-hpa-axis-to-mild-blast-traumatic-brain-tDAvno6LlT
SP - 1
EP - 2375
VL - Advance Article
IS - 6
DP - DeepDyve
ER -