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The dynorphin/kappa opioid receptor mediates adverse immunological and behavioral outcomes induced by repetitive blast trauma

Abstract

Background

Adverse pathophysiological and behavioral outcomes related to mild traumatic brain injury (mTBI), posttraumatic stress disorder (PTSD), and chronic pain are common following blast exposure and contribute to decreased quality of life, but underlying mechanisms and prophylactic/treatment options remain limited. The dynorphin/kappa opioid receptor (KOR) system helps regulate behavioral and inflammatory responses to stress and injury; however, it has yet to be investigated as a potential mechanism in either humans or animals exposed to blast. We hypothesized that blast-induced KOR activation mediates adverse outcomes related to inflammation and affective behavioral response.

Methods

C57Bl/6 adult male mice were singly or repeatedly exposed to either sham (anesthesia only) or blast delivered by a pneumatic shock tube. The selective KOR antagonist norBNI or vehicle (saline) was administered 72 h prior to repetitive blast or sham exposure. Serum and brain were collected 10 min or 4 h post-exposure for dynorphin A-like immunoreactivity and cytokine measurements, respectively. At 1-month post-exposure, mice were tested in a series of behavioral assays related to adverse outcomes reported by humans with blast trauma.

Results

Repetitive but not single blast exposure resulted in increased brain dynorphin A-like immunoreactivity. norBNI pretreatment blocked or significantly reduced blast-induced increase in serum and brain cytokines, including IL-6, at 4 h post exposure and aversive/anxiety-like behavioral dysfunction at 1-month post-exposure.

Conclusions

Our findings demonstrate a previously unreported role for the dynorphin/KOR system as a mediator of biochemical and behavioral dysfunction following repetitive blast exposure and highlight this system as a potential prophylactic/therapeutic treatment target.

Introduction

Mild traumatic brain injury (mTBI) is a serious public health issue for Veterans and military Servicemembers (SM). Moreover, proliferating use of high explosives against civilian populations in regions of conflict pose growing risks of blast-related mTBI globally. Approximately 75–90% of all traumatic brain injuries in the United States are mild [18, 51, 78]. Often referred to as the “signature injury” in Veterans and SMs from the Iraq/Afghanistan wars (OIF/OEF/OND) [52], mTBI is reported at higher incidence in SMs compared to the civilian population [61]. While approximately 10–20% of individuals from OEF/OIF/OND have experienced at least one TBI [36, 72], 75–85% of all TBIs reported are from repetitive blast exposure [27, 74]. Likewise, combat training, terrorist bombings, and industrial accidents are also commonly associated with blast trauma [63]. While health issues can resolve within several weeks, significant persisting symptoms (cognitive, affective, and pain interference) can decrease quality of life and daily functioning [3, 45]. Critically, preventive measures and treatment options for these chronic and debilitating symptoms remain limited.

Mechanisms underlying adverse outcomes following repetitive blast exposure are not understood and are an area of active investigation. Roughly 40% of Veterans from OIF/OEF/OND with a history of blast exposure met criteria for polytrauma (e.g., the Polytrauma Clinical Triad), consisting of post concussive symptoms from mTBI, posttraumatic stress disorder (PTSD), and chronic pain [35]. Results from animal studies also demonstrate comorbid outcomes related to polytrauma following blast exposure. We and others have reported on similar behavioral phenotypes in rodents following blast exposure as is present in SMs and Veterans with a history of blast trauma, including disinhibition and risk taking [68], executive dysfunction [5], substance use disorder risk [67], and aversion/increased stress-reactivity [20, 55, 69].

One potential molecular mechanism capable of contributing to both mTBI, PTSD, and chronic pain-related outcomes is activation of the dynorphin/kappa opioid receptor (KOR) system. Indeed, this receptor system has been implicated in adverse outcomes following mild to severe head impact trauma [6, 25, 58], but has not been investigated in a blast trauma setting (mild or severe). KORs are expressed widely throughout the brain and dynorphin/KOR activation is responsible for the dysphoric/aversive component of stress and pain [8, 10, 34]. Likewise, mild blast trauma is associated with hypothalamic–pituitary–adrenal (HPA) axis dysfunction [77] and KORs are expressed within the HPA axis, where they help regulate stress response and neuroendocrine function [76]. Cortisol levels are acutely increased in human males post blast exposure [14] and we demonstrated a similar blast-dose effect on increased corticosterone release in male mice [77]. In that study, we also demonstrated that repetitive but not single blast exposure results in aversion to blast-paired cues, highlighting the potential for dynorphin/KOR to play a crucial role in adverse blast outcomes.

Dysregulation of the immune system is also a potential mechanism underlying adverse blast outcomes and may interact with the dynorphin/KOR system. Both TBI and PTSD alone can result in increased production of inflammatory cytokines, such as IL-6 and TNF-α [19, 71, 81]. Likewise, in SMs and Veterans with comorbid PTSD and TBI, IL-6 increase is associated with symptom severity [23, 62] and is considered a therapeutic target of interest [48]. In addition, HPA axis dysfunction in mTBI may be linked to altered pro- and anti-inflammatory responses [73]. KORs are expressed on immune cells [40], can influence IL-6 and TNF-α levels [4], and help mediate aspects of acute and chronic neuropathic pain [10, 37, 42]. While previous results have demonstrated acute dynorphin increase following mild to severe impact-induced brain injury in cats and rodents [32, 44, 60], the dynorphin/KOR system has not been studied in blast-exposed individuals nor in animal blast models.

These considerations lead us to hypothesize that repetitive mild blast exposure results in dynorphin level increase and subsequent activation of the KOR system to mediate adverse outcomes following blast exposure. To test this, we utilized our well-established mouse model of blast that accurately recapitulates open-field blast forces [5, 38, 39, 46, 69, 68], in combination with established dynorphin A-like immunoreactivity and cytokine assays and behavioral tests related to adverse blast outcomes. Building on our finding of increased serum corticosterone acutely following blast exposure [77], here we show that repetitive but not single blast exposure resulted in an acute increase in brain dynorphin A-like immunoreactivity of male mice. We then tested the ability of a KOR antagonist to block and/or reduce adverse blast outcomes by pre-treating male mice with nor-binaltophimine (norBNI), a selective long-acting (receptor inactivating) KOR-antagonist lasting up to 21 days in rodents [9, 8, 7, 16, 66]. Pretreatment with norBNI prior to repetitive blast exposure blocked some, but not all, acute cytokine changes in both blood serum and brain. Importantly, we further demonstrate that KOR antagonism completely blocked aversive- and anxiety-like behavioral outcomes and blast-induced light aversion but not motor incoordination. Taken together, these data suggest that the dynorphin/KOR system mediates adverse blast-induced inflammatory response and behavioral affective regulation.

Materials and methods

Animals

Male wild-type C57Bl/6 mice (Jackson Laboratory) 9 weeks of age upon arrival were given 1 week to acclimate, followed by an additional week of handling habituation prior to sham or blast exposure. Animals are randomly assigned by cage (3 animals per cage) to vehicle, drug, sham, or blast groups. Animals were group-housed with access to food and water ad-libitum on a 12:12 light:dark cycle (lights on at 6 am), with the exception of during training for the photophobia assay, where their food was restricted to maintain 83–90% of their ad libitum body weight. All animal experiments were conducted in accordance with Association for Assessment and Accreditation of Laboratory Animal Care guidelines and were approved by the VA Puget Sound Institutional Animal Care and Use Committee.

KOR antagonist pretreatment

Three days prior to sham/blast exposure, animals were injected intraperitoneally with either 10 mg/kg nor-binaltorphimine dihydrochloride (EMD Millipore, Burlington, MA) dissolved in sterile saline or vehicle (saline; 10 mL/kg).

Mouse model of blast trauma

The shock tube (Baker Engineering and Risk Consultants, San Antonio, TX) was designed to generate blast overpressures (BOP) that mimic open field high explosive detonations experienced by military SMs in the Iraq and Afghanistan wars. Design and modeling characteristics have been described in detail elsewhere [30, 68]. Briefly, mice were anesthetized with isoflurane (induced at 5% and maintained at 2%), secured to gurney, and then placed into shock tubes, where the ventral body surface was facing the oncoming shock wave [33]. Sham control animals received anesthesia only (same room and induction chamber as for blast exposure) for a duration matched to blast animals. Anesthesia duration ranged from 3 to 5 min. Animals were exposed to sham/blast exposure either once (1x) or over the course of 3 days, one per day (3x). Following exposure, mice were immediately removed from the shock tube, anesthesia discontinued, and the mouse placed on its back to determine loss of righting reflex (LORR) time (amount of time required for the mouse to flip over and place hind legs 2x). Average BOP peak intensity (psi) was in keeping with mild blast TBI (19.59 psi ± 1.41) [33, 13]. Under these experimental conditions, the overall survival rate is 97%, with blast-exposed mice comparable to sham-exposed mice upon inspection 2–4 h following exposure (e.g., responsive to stimuli, normal posture, and breathing). Animals were weighed on each day of blast/exposure, 24-, 48-h, and 1-month post-exposure. Sham 1 × and 3 × animals were pooled together as there were no statistically significant differences for the dynorphin immunoassay.

Enzyme-linked immunosorbent assay, immunoassays

Cytokine

Animals (n's: sham vehicle: 8, sham norBNI: 12, blast vehicle: 9, blast norBNI: 12) were sacrificed 4 h following final sham/blast exposure and trunk blood and brain collected for downstream biochemical analysis (Fig. 1A). Serum was collected in a serum separator gel tube and was allowed to clot at room temperature for 30–45 min before centrifugation (3000×g) for 10 min. Brains were placed in a 2.0 mL BeadBug pre-filled microcentrifuge tube on ice containing 1.0 mm acid washed silica beads (Sigma, Burlington, MA) and 1 mL of total lysis buffer solution. Lysis buffer consisted of 0.02% Triton-X, 10 mM HEPES, 1.5 mM MgCl2, and 10 mM KCl, with fresh protease/phosphatase inhibitor cocktails (Sigma, Burlington, MA) [21]. Brains in ice cold lysis buffer were then homogenized twice with a BeadBug (Sigma, Burlington, MA) at 4000 rpm for 45 s each. The samples were then centrifuged at 4 °C at 18,000×g for 10 min and the supernatant was aliquoted and stored at − 80 °C. Pro- and anti-inflammatory cytokine level were then analyzed using the IDEXX Bioanalytics Mouse Multiplex Cytokine Panel (23-plex) and service (samples shipped to IDEXX on dry ice overnight). To normalize variable protein amounts, brain lysates were assayed for total protein concentration using Pierce BCA protein assay (Thermo Fisher Scientific, Rockford, Illinois) as per manufacturer protocol (50-fold dilution). Measurements were completed by reading absorbance at 562 nm.

Fig. 1
figure 1

Timeline and acute effects of blast trauma. A Master timeline of experimental study. B Dynorphin A-like immunoreactivity is increased in the brain after 3 × but not 1 × blast. One-way ANOVA. Tukey’s post-hoc analysis. C norBNI does not block acute weight loss. Two-way ANOVA. Bonferroni–Šídák post-hoc analysis. D norBNI does not block LORR delay. Two-way RM ANOVA. Tukey’s post-hoc analysis

Dynorphin

Animals (n's: sham: 8, blast 1×: 5, sham 3×: 4) were sacrificed 10 min following final sham/blast exposure brain collected for downstream biochemical analysis (Fig. 1A). Brains were homogenized with ice cold lysis buffer as above. Dynorphin A-like immunoreactivity was assayed using a Dynorphin A enzyme immunoassay (EIA) kit (Raybiotech, Peachtree Corners, GA) as per manufacturer protocol (16-fold dilution) [17, 59]. The assay is a quantitative competition-based ELISA on a 96-well strip plate and Dynorphin A-like immunoreactivity is determined using a standard curve. Detection range: 0.1–1000 ng/mL; sensitivity: 0.3 ng/mL. Measurements were completed by reading absorbance at 450 nm.

Behavioral battery

Behavioral testing was conducted 1-month post-sham/blast exposure and consisted of two testing batteries each conducted in separate groups of mice over the course of 1 week (one test paradigm per day; Fig. 1A). Each mouse only completed one of the testing batteries as follows: (1) marble burying, place conditioning, acoustic startle or (2) rotarod, odorant conditioning, photophobia reward conflict assay). The order of behavioral tests for each battery was performed from least to most stressful to decrease carryover distress from one behavior to the next. Prior to each day of testing, animals were placed in the testing room for at least 30 min to allow for acclimation. Cohort 1 n’s: sham vehicle: 17, sham norBNI: 19, blast vehicle: 17, blast norBNI: 15; Cohort 2 n’s: sham vehicle: 14, sham norBNI: 15, blast vehicle: 15, blast norBNI: 12.

Cohort 1 (Fig. 4A)

Marble burying

Animals were allowed to explore an open field in a clean rat cage, 259 mm × 476 mm × 209 mm, 909 cm2 floor space) filled with 5 cm of bedding (kernels) and 18 marbles for 30 min. Marbles were counted as buried if at least two-thirds of the height of the marble was covered with bedding.

Place conditioning paradigm

A balanced-three compartment conditioning apparatus was used as described previously [69]. One week prior to sham/blast exposure, animals were pre-tested by placing individual animals in the small central compartment and allowed exploration of the entire apparatus for 20 min. Mice were then counterbalanced to gray or vertical black and white stripes for their morning pairing based on initial side bias. Animals showing side bias were excluded. Sham/blast exposure occurred the following week over three consecutive days. On each day of exposure, in the morning, animals were placed in their AM-pairing chamber containing distinct visual cues for 10 min and then were immediately given a sham exposure. In the afternoon of the same day, animals were placed in their PM-pairing chamber containing a different set of distinct visual cues for 10 min and then were immediately given a blast or sham exposure (depending on group assignment). Place conditioning was assessed at 1 month following exposure by allowing each mouse to roam freely in all three compartments. Time spent in each compartment was recorded and analyzed using Anymaze (Stoelting, Wood Dale, IL). Place conditioning scores were calculated by subtracting time spent in the PM paired compartment from time spent in the AM paired compartment.

Acoustic startle

Conducted using SR-LAB acoustic startle boxes (San Diego Instruments, San Diego CA). Following a 5-min acclimation period, startle habituation testing consisted of 50 trials of 120-dB pulses delivered with an inter-trial interval of 7–23 s. Blast exposure can result in hearing loss in rodents [41], which also occurs in blast-exposed Veterans [53]. Therefore, we use the within subject analysis approaches of startle habituation in an attempt to mitigate potential confounds of hearing loss on startle outcome measures and interpretation. Startle habituation was expressed as the ratio of the average startle response in the last 10 trials to the average startle response in the first 10 trials (1.0 = no habituation).

Cohort 2 (Fig. 5A)

Rotarod

A rotarod apparatus (San Diego Instruments, San Diego, CA) was used to measure motor coordination and balance. The behavior was assessed over 2 days: acclimation and test day. On the acclimation day, three trials were used at the speed of 16 rpm. On the test day, five trials were used, each with a different set speed of rotation (in order of 8, 16, 24, 32, and 40 rpm). For each trial, the rod would accelerate to a set speed over 120 s, remain at the speed for an additional 30 s, and finally decelerate back to 0 rpm over 30 s. The latency to fall off the rotarod was recorded for each trial and area under the curve (AUC) across all five trials was calculated.

Odorant conditioning paradigm

ON each day of sham/blast exposure, animals received a stainless-steel mesh tea-ball (Amazon) containing one quarter Nestletts (PharmaServ, Framingham, MA) with 30 μl of imitation almond extract (Kroger, Cincinnati, OH) in their home cage 5 min prior to sham/blast exposure. The Nestletts and scents were refreshed on each subsequent sham/blast exposure and the tea-ball remained in place until 24 h following final sham/blast exposure. One month following exposure, animals were tested for odorant-conditioning in a Plexiglas T-maze (66 cm long × 40 cm wide × 15 cm high) with a tea-ball containing one-quarter Nestlett with 30 μl imitation almond odorant cue placed in the left arm of the maze and a tea-ball containing one-quarter Nestlett with no odor placed in the opposite arm of the maze. Animals were placed in the long arm of the T-maze and given 5 min to explore the entire maze. Distance traveled and time spent in each of the two distal ends of the short arms were recorded and analyzed using Anymaze (Stoelting, Wood Dale, IL). Aversion is calculated as (scent/(scent + no scent).

Photophobia reward conflict assay

The photophobia assay was conducted in an Orofacial Pain Assessment Device (OPAD) using Anymaze acquisition and analysis software (Stoelting, Wood Dale, IL), and consisted of a choice between self-administration of a reward vs. exposure to a bright light. Mice were food-restricted 1 week prior and throughout the duration of testing. To decrease neophobia during training, mice were given ad libitum access to a sweetened condensed milk solution (1:2 solution of sweetened condensed milk in water (autoclaved, obtained from the animal facility) in their home cage for 24 h. Animals were then trained to self-administer the milk solution from a sipper tube over 6 sessions (one session per day) of 9 min each. A lamp was positioned over the milk bottle but the light was not turned on during training sessions. The metal sipper tube with milk solution is attached to a stand and placed in a position, where the spout is accessible to rodent, which enables the recording of individual licks as well as total lick duration. The general room lighting was maintained at 85 lx throughout. Testing occurred on the 7th session and consisted of an initial 3 min of no light followed by 3 min of a bright light (1000 lx) turned on directly above the sipper tube location, and then a final 3 min with no light. Photophobia ratio was calculated by dividing the duration of licking during the 3 min of light on by the duration of licking during the initial 3 min with no light.

Data analysis

As appropriate, data were analyzed using: (i) standard one-way analysis of variance (ANOVA), followed by Tukey’s Post-hoc tests; (ii) two-way (between/within subjects design) ANOVA, followed by Bonferroni–Šídák or Tukey’s Post-hoc tests (repeated measures (RM) where appropriate). Reported significant p values denote two-tailed probabilities of p ≤ 0.05 and non-significance (n.s.) indicates p > 0.05. Data were excluded in behavioral testing if the data point was equal to or greater than two standard deviations from the group mean. Statistical analyses were conducted using custom Python applications and Graph Pad Prism 4.0 (GraphPad Software, Inc., La Jolla, CA).

Results

Using well-established blast methods [5, 38, 39, 46, 77,78,67], male C57Bl/6 adult mice were exposed to one (1x) or three (3x) blast BOPs using a pneumatic shock tube delivering a peak static pressure of 19.59 ± 1.41 psi. Sham control mice were anesthetized equivalently to blast-exposed animals (see Materials and methods). Figure 1A represents a general timeline of experiments performed in this study. Additional detail regarding cohorts undergoing each behavioral battery is shown in Figs. 4A and 5A.

Repetitive but not single blast exposure increases brain dynorphin A-like immunoreactivity, but KOR antagonism does not prevent blast-induced loss of righting reflex or acute weight loss

Corticosterone levels are increased in rodents after blast exposure [69, 82] and male mice demonstrate blast-induced aversion [69], highlighting a potential role for the dynorphin/KOR system. We first tested whether single or repetitive (3X) blast exposure (one exposure per day) resulted in changes to brain dynorphin A-like immunoreactivity levels using an immunoassay in whole brain lysate. We found that repetitive, but not single, blast exposure was necessary to elicit increased whole brain dynorphin A-like immunoreactivity levels 10 min post blast-exposure (one-way ANOVA: F[2, 14] = 5.146, p = 0.021, Tukey’s; n = 5–8) (Fig. 1B).

Since dynorphin has been associated with stress exposure, we next investigated whether the long-acting receptor-inactivating KOR antagonist, norBNI, affects acute outcomes following sham or blast exposure. norBNI and saline vehicle was systemically administered to both blast and sham mice (see Fig. 1A) 3 days prior to exposure. On exposure days, we measured weight change and LORR immediately after sham/blast as measures of acute physical stress response. We found that norBNI did not prevent weight loss 24-h post-blast (two-way ANOVA: interaction effect F[1, 86] = 1.042, p = 0.310, main effect of treatment F[1, 86] = 330.4, p < 0.0001, main effect of drug F[1, 86] = 0.759, p = 0.386, Bonferroni–Šídák; n = 21–24) (Fig. 1C) nor prevent the blast-induced LORR (two-way RM ANOVA: time × treatment effect F[6,172]  = 2.480, p = 0.025, main effect of group F[3, 86] = 69.00, p < 0.0001, main effect of time F[1.911,171.2] = 1.976, p = 0.142, Tukey’s; n = 21–24) (Fig. 1D).

KOR antagonism blocks most cytokines acutely after repetitive blast exposure

Since cytokine release is commonly associated with blast trauma [23, 64] and dynorphin/KOR plays a role in inflammation and negative affect [4, 8, 80], we next tested whether KOR antagonism at the time of blast exposure blocks blast-induced cytokine changes in both brain and blood serum collected 4 h post-exposure using an established 23-plex immunoassay (Fig. 1A. The 23-plex assays for IL-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12(p40, IL-12(p70, IL-13, IL-15, IL-17, G-CSF, GM-CSF, INF-y, IP-10, MKC, MCP-1, MIP-1a, MIP-1b, MIP-2, RANTES, TNFa. A subset of the 23 analytes fell below the level of detection (detailed below and were excluded from further analysis. These data are in agreement with the mild nature of our blast exposure paradigm as previously reported [23, 64]. All statistics for this assay are detailed in Tables 1 and 2 for serum and brain, respectively. All analyses used two-way ANOVAs and post-hoc Bonferroni–Šídák.

Table 1 Statistical analysis of cytokine levels in blood serum
Table 2 Statistical analysis of cytokine levels in brain lysate

In serum, only seven cytokines were detected at appreciable levels: IL-1α, IL-5, IL-6, G-CSF, IP-10, MKC (CXCL1), and MCP-1. norBNI pretreatment had differential effects on specific cytokines in the serum. Neither repetitive blast exposure nor KOR antagonist treatment altered IL-1α levels (Fig. 2A). Repetitive blast exposure significantly increased IL-5, IL-6, G-CSF, IP-10 and MKC in blast when compared to the sham controls, and norBNI attenuated those effects (Fig. 2B–F). norBNI did not affect blast-induced MCP-1 increase (Fig. 2G).

Fig. 2
figure 2

norBNI pretreatment blocked some but not all inflammatory cytokines in blood serum. norBNI nor blast exposure alter A IL-1α levels. norBNI attenuated blast-induced B IL-5, C IL-6, and F levels. norBNI did not block blast-induced D G-CSF and E IP-10 levels and there is no difference between norBNI groups. G norBNI did not block blast-induced MCP-1 levels. AG Two-way ANOVA. Bonferroni–Šídák post-hoc analysis. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns: not significant. Values represent mean ± SEM

In brain lysates, 12 cytokines were detected: IL-1α, IL-6, IL-9, IL-15, IL-17, G-CSF, INF-γ, IP-10, MKC, MCP-1, and RANTES (Fig. 3A–L). Compared to serum, blast and norBNI effects were more robust and consistent in the brain. All cytokines detected except IL-15 (Fig. 3E) and MKC (Fig. 3J) had significantly increased levels in the blast vehicle group as compared to the sham vehicle group (trend for increase in IL-15 following blast). When compared to the vehicle blast group, norBNI blocked all blast-induced changes except for MKC (Fig. 3J). Of the cytokines analyzed, IL-6 was the only cytokine whose increase following repetitive blast exposure was blocked by norBNI in both serum and brain.

Fig. 3
figure 3

norBNI pretreatment blocked most but not all inflammatory cytokines in the brain. norBNI blocks blast-induced released in A IL-1a, B IL-6, C IL-9, D IL-13, E IP-15, G G-CSF, H INF-y, I IP-10, K MCP-1, and L RANTES, but not in F IL-17. J MKC levels are not affected by blast nor norBNI. E IP-15 does not increase in the vehicle blast group. AL Two-way ANOVA. Bonferroni–Šídák post-hoc analysis. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns: not significant. Values represent mean ± SEM

KOR antagonism blocks repetitive blast-induced anxiety-/aversive-like behavior and photosensitivity but not ambulatory deficit

Separate sets of mice were tested in two sets of behavioral measures. In the first group of mice, a discrete environmental stimulus was presented for 10 min prior to sham/blast exposure. Behavioral testing then occurred 1 month following repetitive sham or blast exposure and included marble burying (anxiety/compulsivity), place conditioning (aversion), and acoustic startle (habituation) (Fig. 4A). norBNI pretreatment blocked aversion to paired visual cues (two-way ANOVA: interaction effect F[1, 41] = 2.499, p = 0.122, main effect of treatment F[1, 41] = 6.943, p = 0.012, main effect of drug F[1, 41] = 3.667, p = 0.063, Bonferroni–Šídák; n = 10–12) (Fig. 4B), and did not have an effect on distance traveled (two-way ANOVA: interaction effect F[1, 41] = 0.055, p = 0.815, main effect of treatment F[1, 41] = 0.577, p = 0.452, main effect of drug F[1, 41] = 1.251, p = 0.270, Bonferroni–Šídák; n = 10–12) (Fig. 4C). Therefore, differences in time spent in the blast-paired environment were not attributable to locomotor differences. Likewise, norBNI blocked the blast-induced increased in number of marbles buried (two-way ANOVA: interaction effect F[1, 64] = 4.580, p = 0.036, main effect of treatment F[1, 64] = 2.370, p = 0.129, main effect of drug F[1, 64] = 1.007, p = 0.319, Bonferroni–Šídák; n = 15–19) (Fig. 4D) and inhibited acoustic startle habituation (two-way ANOVA: interaction effect F[1, 51] = 6.194, p = 0.016, main effect of treatment F[1, 51] = 2.643, p = 0.11, main effect of drug F[1, 51] = 2.161, p = 0.148, Bonferroni–Šídák; n = 12–15) (Fig. 4E).

Fig. 4
figure 4

norBNI blocks blast effect in anxiety- and aversive-like behavior in PCP. A Timeline of behavioral battery of cohorts undergoing PCP. B norBNI blocks aversive-like behavior in PCP. C norBNI does not block locomotion in PCP. D norBNI blocks blast effect in number of marbles buried. E norBNI blocks delayed habituation rate blast effect in acoustic startle response. BE Two-way ANOVA. Bonferroni–Šídák post-hoc analysis. *p ≤ 0.05, **p ≤ 0.01, ns: not significant. Values represent mean ± SEM

For the second group of mice, sham/blast exposure was paired with a liquid almond extract liquid absorbed onto a Nestlet. The Nestlet with almond extract was enclosed in a tea ball strainer and placed in the cage for 5 min before sham or blast exposures (Fig. 5A). One month following repetitive sham or blast exposure, mice were tested in order listed: OCP (aversion), rotarod (motor coordination), and the photophobia reward conflict (photosensitivity) as sensitivity to light has been reported in blast TBI patients [11, 75]. In line with results from the place conditioning experiments, norBNI blocked aversion to blast-paired odor (two-way ANOVA: interaction effect F[1, 50] = 4.160, p = 0.0.047, main effect of treatment F[1, 50] = 4.214, p = 0.043, main effect of drug F[1, 50] = 0.111, p = 0.740, Bonferroni–Šídák; n = 12–16) (Fig. 5B) but did not affect distance traveled (two-way ANOVA: interaction effect F[1, 50] = 0.473, p = 0.495, main effect of treatment F[1, 50] = 0.328, p = 0.569, main effect of drug F[1, 50] = 0.291, p = 0.592, Bonferroni–Šídák; n = 12–16) (Fig. 5C). Conversely, norBNI did not block or decrease blast-induced deficits on the accelerating rotarod (two-way ANOVA: interaction effect F[1, 74] = 2.419, p = 0.124, main effect of treatment F[1, 74] = 23.91, p < 0.0001, main effect of drug F[1, 74] = 1.031, p = 0.313, Bonferroni–Šídák; n = 19–20) (Fig. 5D). Following these behaviors, mice had their food access restricted and were given access to a sweetened condensed milk solution in a modified OPAD for 9 min per day (see Methods). On the final test day, a bright light was lit directly over the access point to the milk solution at the 6-min mark. Remarkably, we found norBNI blocked increased blast-induced aversion to light (two-way ANOVA: interaction effect F[1, 50] = 6.733, p = 0.012, main effect of treatment F[1, 50] = 0.562, p = 0.457, main effect of drug F[1, 50] = 0.767, p = 0.385, Bonferroni–Šídák; n = 12–15) (Fig. 5E).

Fig. 5
figure 5

norBNI blocks blast effect in light sensitivity- and aversive-like behavior in OCP. A Timeline of behavioral battery of cohorts undergoing OCP. B norBNI blocks aversive-like behavior in OCP. C norBNI does not block locomotion in OCP. D norBNI does not block blast effect in latency to fall in rotarod. E norBNI blocks blast effect in time spent away from light. BE Two-way ANOVA. Bonferroni–Šídák post-hoc analysis. *p ≤ 0.05, **p ≤ 0.01, ns: not significant. Values represent mean ± SEM

Discussion

In this study, we uncovered a critical role for repetitive blast-induced dynorphin/KOR activation in mediating discrete aspects of (neuro)-inflammation and behavioral dysfunction at acute and chronic timepoints in male mice. The majority of all TBIs in the US are mild [18, 51, 78] and can result in significant adverse outcomes and decreased quality of life. However, currently there are no approved prophylactic or therapeutic treatment approaches for adverse outcomes following blast TBI. While the dynorphin/KOR system has been implicated in severe impact TBI [32, 44, 60], there has been no investigation of this receptor system in models of impact or blast mTBI. Here, we present novel evidence for an increase in brain-wide dynorphin A-like immunoreactivity acutely following repeated, but not single blast exposure. These results are consistent with previous findings that demonstrate only repetitive blast exposure resulted in aversion to blast-paired cues [69]. Furthermore, here we extend on our previous finding by showing that systemic administration of the selective long-lasting KOR antagonist norBNI blocked or decreased blast-induced cytokine changes in serum and brain samples and discrete aspects of chronic behavioral dysfunction. Together these results highlight the dynorphin/KOR system as a mediator of blast-induced dysfunction and support further research aimed at establishing this system as a potential prophylactic and/or therapeutic target for adverse outcomes following repetitive blast trauma.

In this study, we focused our efforts on norBNI, a selective long-acting KOR-antagonist lasting up to 21 days in rodents [11,12,7, 16, 66]. While administration of a KOR antagonist had effects on acute cytokine levels and chronic behavioral dysfunction, KOR antagonism did not block LORR immediately following blast exposure nor acute weight loss, suggesting that these adverse outcomes are mediated by other systems and processes. Likewise, KOR antagonism blocked or altered acute cytokine changes following repetitive blast exposure in the brain more robustly than in blood serum. Together, these results demonstrate the ability of this pharmacological approach to isolate and link discrete pathophysiological and behavioral outcomes associated with mTBI, which can in turn inform downstream translation and precision medicine efforts.

Pretreatment with norBNI blocked blast-induced IL-6 increase, which may offer neuroprotection in acute injury but has also been correlated with health risks chronically in both TBI and PTSD [48]. The differential effect between the brain and serum cytokine levels may be due to alterations of blood–brain barrier (BBB) permeability or differing dynorphin/KOR mechanisms in the periphery versus the brain [6, 8, 10]. While there is a lack of research investigating dynorphin/KOR involvement in BBB permeability, KOR may be implicated in BBB integrity through its actions on the HPA axis and/or inflammatory response. Despite having acutely increased cortisol [14], SMs have below baseline levels of cortisol at more chronic timepoints [79] and TBI [2]. In addition, glucocorticoids may play an important role for BBB recovery in blast trauma by tightening the barrier resulting in decreased peripheral immune invasion and inflammation [31]. Furthermore, blast-induced activation of the dynorphin/KOR system may influence (neuro)-inflammation and BBB dysfunction through priming stress-stimulated immune cells [65, 73]. Importantly, systemic administration of norBNI can pass the BBB [50, 54]; therefore, KOR antagonism may provide protective benefits via mechanisms outside of BBB disruption and/or systemic inflammation.

Moving forward, a detailed examination of dynorphin and cytokine changes post blast in discrete brain regions (e.g., amygdala, cortex, striatum, cerebellum) is warranted as results here detail whole brain levels, which may mask more discrete changes within specific brain regions. These efforts are ongoing and in parallel with immunohistochemical efforts to understand a potential role for dynorphin/KOR in blast-induced microglia and astrocyte dysfunction. Our results demonstrating an effect of systemic KOR antagonism on adverse blast outcomes suggest that repetitive blast trauma results in release of dynorphin and activation of KORs. Conversely, it is important to note that the current study did not directly assess dynorphin release, so while unlikely, the increase in dynorphin A-like immunoreactivity seen here could alternatively represent increased production and storage of dynorphin in intracellular vesicles. Experiments using subcellular fractionation techniques to isolate dense core vesicles (vesicles which release dynorphin peptide from neurons) and/or in vivo imaging of a genetically encoded dynorphin sensor [1] could also increase understanding of how and where blast exposure impacts dynorphin release and cytokine expression. Likewise, mass-spectrometric examination of prodynorphin gene products in extracellular fluid pre/post-blast might also lend important information regarding how blast trauma affects prodynorphin processing and release.

Depression and anxiety are common behavioral symptoms associated with blast trauma in Veterans and SMs, and there is ongoing debate regarding specific contributions of blast-induced mTBI vs. PTSD to these long-term outcomes [26]. Here, we provide evidence that the dynorphin/KOR system encodes aversive- and anxiety-like components of blast trauma. We also find that KOR antagonism blocked aversive-like behavior in a photophobia assay, suggesting this adverse outcome may be stress-mediated and associated with negative pain affect [10]. Conversely, here we did not find an effect of the dynorphin/KOR system on motor coordination skills. This is consistent with the proposed role of the dynorphin/KOR system in primarily mediating affective components of stress and trauma exposure [24, 76]. As rotarod requires skill in balance, grip strength, and motor coordination, mechanisms outside of the dynorphin/KOR system may be mediating these effects [49]. We have previously demonstrated a blast-induced decrease in rotarod performance that is mediated by nitric oxide synthase-dependent BBB permeability [38, 39], and a relationship between dynorphin and nitric oxide has been previously suggested [28, 70], but a role for dynorphin/KOR in BBB permeability nor NOS function post repetitive blast has not been examined.

Our results underscore the need for further investigation of the dynorphin/KOR system’s role in blast-induced trauma, including executive dysfunction and chronic pain. The dynorphin/KOR system interfaces with mesolimbic circuitry and can regulate dopamine dynamics, and we have previously reported aberrant phasic dopamine release in the nucleus accumbens and executive dysfunction in both mice and Veterans with blast mTBI [5, 22, 68]. Therefore, future directions should include investigation of dynorphin/KOR’s involvement in blast-induced dopamine dysfunction and adverse behavioral outcomes related to executive function. In addition, the dynorphin/KOR system has been implicated in drug reward and substance use disorder, which is increased in SMs and Veterans following blast trauma and in mouse models of blast [47, 67]. Finally, we recognize the importance of assessing the potential contribution of sex as a biological variable in mechanisms underlying blast trauma outcomes [43] as sex differences in dynorphin/KOR function have been previously reported [15] and combat military occupational specialties are now available not only to males. To further increase translational relevance, additional blast modeling efforts are also warranted as Servicemembers’ blast exposure experience can vary widely in number of blast exposures (1–100 s have been previously reported) and/or intervals between blasts (e.g., multiple blasts over a day or multiple days vs. multiple blasts from multiples deployments) [29, 56, 57].

Conclusions

This study demonstrates a critical role for the dynorphin/KOR system in mediating discrete aspects of (neuro)-inflammation and behavioral dysfunction at acute and chronic timepoints following repetitive blast trauma in male mice. These results highlight the dynorphin/KOR system as a promising prophylactic and therapeutic target for overlapping comorbidities resulting from repetitive blast trauma and demonstrate KOR antagonists as a tool for increased mechanistic understanding of blast-induced psychopathology. While an estimated 400,000 Veterans have experienced blast mTBI [18, 27, 72], to date there are currently no approved pharmacological agents for prevention/treatment of adverse outcomes. KOR antagonists are currently under clinical trial investigation in the civilian population for treatment of non-blast stress-related disorders [12], but the potential of these drugs in a blast setting has not been investigated. Therefore, increased research devoted to prevention/treatment efforts with KOR antagonists in animal models and in clinical settings is warranted and has a high likelihood of success.

Availability of data and materials

The data in this study are available from the corresponding author upon reasonable request. The manuscript has been uploaded to bioRxiv preprint: https://doi.org/10.1101/2022.08.15.504055

Abbreviations

ANOVA:

Analysis of variance

BBB:

Blood–brain barrier

BOP:

Blast overpressure

HPA:

Hypothalamic–pituitary–adrenal

KOR:

Kappa opioid receptor

LORR:

Loss of righting reflex

mTBI:

Mild traumatic brain injury

norBNI:

Nor-binaltorphimine dihydrochloride

OCP:

Odorant conditioning paradigm

OIF/OEF/OND:

Operation Iraqi Freedom/Operation Enduring Freedom/Operation New Dawn

OPAD:

Orofacial Pain Assessment Device

PCP:

Place conditioning paradigm

PTSD:

Posttraumatic stress disorder

SM:

Servicemember

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Acknowledgements

We would like to thank Scott Ng Evans, Traci J. Weber, Cindy Pekow, DVM, Kari Koszdin, DVM, Monica, and Lena Strait-Bodeyfor considerable technical assistance and veterinary care.

Disclaimer

The views expressed in this scientific presentation are those of the author(s) and do not reflect the official policy or position of the U.S. government or Department of Veteran Affairs.

Funding

This work was supported by grants from NIDA Training Grant 2T32DA007278-26 (BMB) a Department of Veteran Affairs (VA) Basic Laboratory Research and Development (BLR&D) Career Development Award 1IK2BX003258 (AGS), a VA BLR&D Merit Review Award 1I01BX005582 (AGS), a VA BLR&D Merit Review Award 5I01BX002311 (DGC), University of Washington Friends of Alzheimer's Research (DGC), and the UW Royalty Research Fund (DGC).

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The work presented here was carried out in collaboration among all authors. AS, SJL, AL, and DG contributed to conception and design of the study. SJL, AL, MY, BB, and AS collected and analyzed data. SJL and AS wrote the first draft of the manuscript. All authors contributed to manuscript revision. All authors have seen and approved the manuscript. All authors read and approved the final manuscript.

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Correspondence to Abigail. G. Schindler.

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Lee, S.J., Logsdon, A.F., Yagi, M. et al. The dynorphin/kappa opioid receptor mediates adverse immunological and behavioral outcomes induced by repetitive blast trauma. J Neuroinflammation 19, 288 (2022). https://doi.org/10.1186/s12974-022-02643-3

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Keywords

  • Blast overpressure
  • Mild traumatic brain injury
  • Posttraumatic stress disorder
  • Kappa opioid receptor
  • Dynorphin
  • Cytokine
  • Inflammation