Long-lasting neurobehavioral alterations in burn-injured mice resembling post-traumatic stress disorder in humans

Journal Pre-proof Long-lasting neurobehavioral alterations in burn-injured mice resembling post-traumatic stress disorder in humans

Qing-Hong Zhang, Ji-Wei Hao, Ji Xiao-Jing, Li Guang-Lei, Min Zhou, Yong-Ming Yao PII:

S0014-4886(18)30323-6

DOI:

https://doi.org/10.1016/j.expneurol.2019.113084

Reference:

YEXNR 113084

To appear in:

Experimental Neurology

Received date:

8 August 2018

Revised date:

9 March 2019

Accepted date:

14 October 2019

Please cite this article as: Q.-H. Zhang, J.-W. Hao, J. Xiao-Jing, et al., Long-lasting neurobehavioral alterations in burn-injured mice resembling post-traumatic stress disorder in humans, Experimental Neurology (2018), https://doi.org/10.1016/ j.expneurol.2019.113084

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© 2018 Published by Elsevier.

Journal Pre-proof Long-lasting Neurobehavioral Alterations in Burn-Injured Mice Resembling Post-Traumatic Stress Disorder in Humans

Qing-Hong ZHANG1#*M.D., Ph.D, Ji-Wei HAO1# B.S., Xiao-Jing JI1, 2 M.D., Guang-Lei LI1 M.D., Min Zhou3 M.D., Ph.D, Yong-Ming YAO1* M.D., Ph.D

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Trauma Research Center, Fourth Medical Center of Chinese PLA General Hospital, Beijing 100048,

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P.R.China Department of Emergency, First Hospital Affiliated to Wenzhou Medical College, Wenzhou, Zhejiang,

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Neurocritical care unit, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine,

* Correspondence author: Qing-Hong ZHANG, MD, PhD.

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Yong-Ming YAO, MD, PhD.

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University of Science and Technology of China, Hefei, Anhui, 230001, P.R. China

Trauma Research Center, Fourth Medical Center of Chinese PLA General Hospital, 51 Fucheng Road, Haidian District, Beijing 100048, P.R.China

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3

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325000, P.R. China

Phone: (++86) 1066867382 Fax: (++86) 1068989955

Email: [email protected] [email protected]

# The

two authors contributed equally to the manuscript.

Running title: Long-lasting neurobehavioral alterations in burn mice

This study was supported by National Natural Science Foundation (81272089, 81730057).

Journal Pre-proof Abstract Objective: To establish an animal model for posttraumatic stress disorder in burn-injured patients. Methods: Thermal-injured mice with 15% total body surface area were subjected to a series of neurobehavioral tests at 1 and 3 months postburn. Brains were collected for analysis of key molecules expression, spleens for T cell function analysis, and blood for biochemistry and hormones detection. Results: Comparison with sham mice, burn mice showed extremely high locomotion in homecage, open field, and forced swimming tests, indicating a hyper-arousal state. Burn mice exhibited improved spatial memory in Morris Water Maze test and heightened context fear memory in context fear conditioning,

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suggesting re-experiencing behavior. Although burn mice showed pronounced passive avoidance in the

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step-through test, their active avoidance capability in response to the conditional stimulus in the shuttle

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box test was relatively deteriorated. Likewise, the retention of cue-feared memory was impaired in fear conditioning test. The above negative alterations in mood were recapitulated in open-field test, in which

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the burn mice displayed an anxiety-like behavior with less time spent in the center. However, no sign of

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depression was found in the forced swimming and sucrose preference tests. The negative mood of burn mice was reinforced by a deficit in sociality and preference for social novelty in social interaction test.

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These neurobehavioral alterations were associated with an increased expression of brain-derived neurotrophic factor along with a remarkable microgliosis and a moderate astrocytosis in the brain of burn

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vs. sham mice. Moreover, a prominent Th2 switch and consequent increased nuclear NF-κB translocation were seen in the splenic T cells from burn relative to sham mice. Conclusions: We conclude that even mild burn injury could lead to long-lasting cognitive and effective alterations in mice. These findings shed light on the interactions among neuropsychology, neurobiology, and immunology throughout the recovery period of burn injury.

Keywords: burn injury; neurobehavior; posttraumatic stress disorder; CD4+ T cell; brain-derived neurotrophic factor

Journal Pre-proof Abbreviations posttraumatic stress disorder (PTSD); brain-derived neurotrophic factor (BDNF); central nervous system (CNS); total body surface area (TBSA); Morris Water Maze (MWM); postburn (pb); conditioned stimulus (CS); unconditioned stimulus (US); glial fibrillary acidic protein (GFAP paraformaldehyde (PFA); bovine serum albumin (BSA); polyvinylidene difluoride (PVDF); intraperitoneally (i.p.); ionized calcium binding adaptor protein 1(Iba-1), glial fibrillary acidic protein (GFAP); dopamine receptor (DR); traumatic brain injury (TBI), aspartate transaminase (AST), alanine aminotransferase (ALT), high density lipoprotein

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(HDL), low density lipoprotein (LDL), very low density lipoprotein (VLDL)

Journal Pre-proof 1. Introduction It has been well established that burn injury can increase the risk for mental health issues (Logsetty et al., 2016). Symptoms of posttraumatic stress disorder (PTSD), acute stress disorder, anxiety, depression, itching, delirium, nightmares, and deficits in language and family functioning are commonly experienced in burn patients. Among these, PTSD occurred in approximately 30% of burn injury cases (Wiechman and Patterson, 2004; Rimmer et al., 2014) in the early exposure (Newton et al., 2014) or several years after discharge from hospital (Dahl et al., 2016; Cakir et al., 2015; Sheridan et al., 2014). Characteristic symptoms of PTSD include four major symptom clusters: intrusion (Cluster B), avoidance (Cluster C),

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negative alterations in cognitions and mood (Cluster D), and alterations in arousal and reactivity (Cluster

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E)(Flandreau and Toth, 2018). So far, most of the approaches addressing the burn mental health focus on

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psychology study through epidemic inquisition, with little input from neuroscience-based research. In addition, most of the studies reported an acute neuropathology in an animal model of burn injury (Zhang et

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al., 2011; Reyes et al., 2006; Zhang et al., 2013), whether it evolved into a chronic disease and contributed

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to the neuropsychological abnormality in burn patients remained intriguing. Lastly, although several animal models have been found to successfully model some aspects of PTSD (Deslauriers et al., 2018),

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data on particular symptoms simulating PTSD in burn patient was extremely lacking. Development of valid animal model for PTSD with high translational values in burn injury is critical to elucidate its

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etiology and verify potential treatment efficacy. Accumulating evidence suggested that risk for PTSD be associated with brain-derived neurotrophic factor (BDNF) gene (Felmingham et al., 2013) and its expression (Zhang et al., 2014). In humans, serum BDNF level was positively associated with PTSD; and BDNF-dependent hippocampus plasticity contributed to the greater incidence of PTSD in the adult female rat (Scharfman and MacLusky, 2014). In addition, large evidence for a proinflammatory profile of PTSD and its symptoms was also provided (de la Hoz et al., 2016; Newton et al., 2014; Furtado and Katzman, 2015; Bam et al., 2016b; Bam et al., 2016a). Furthermore, systemic central nervous system (CNS)-specific CD4+ T cells were found to be required for spatial learning and memory and for the expression of BDNF in the dentate gyrus (Radjavi et al., 2014), with the detrimental versus beneficial effects of Th1 cells and Th2 cells, respectively (Gu et al., 2012). At this point, little data exist on the enduring peripheral inflammatory state evoked by burn injury. Since burn injury alone could induce prominent Th2-type shift in adaptive immune reactivity (Guo et al., 2003) along

Journal Pre-proof with alterations in numerous inflammatory cytokines, interleukins and growth factors (Long et al., 2016; Dehne et al., 2002), it might be necessary to re-evaluate T cell immunity in the setting of burn injury to ascertain the long-lasting immune associated mechanism. Elucidating the neurobiological mechanism for PTSD following a moderate burn injury is crucial to accelerate the recovery and effective interventions for psychological distress. We hypothesize that burn injury could induce long-lasting neuropsychological disorder in mice, and the systemic and brain-specific inflammation may account partly for the alterations. Thus, we comprehensively characterized both the cognitive and affective states of burn-survived mice at both 1 and 3 months after thermal injury. Then the

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microglia activation, neurotropic growth factor, pre-synaptic function, and finally the systemic immune

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parameters, were systemically observed.

2. Materials and methods

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2.1 Animals and burn injury model

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Male Balb/c mice (20-25 g, Huafukang Bioscience Co. Inc, Beijing, China) were subjected to dorsal scald thermal or sham injury with 15% total body surface area (TBSA) full thickness as previously

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reported (Zhang et al., 2011). The model was in accordance with the guidelines for humane care of animals by the National Institute of Health in the United States and approved by the Animal Care and Use

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Committee at Chinese PLA General Hospital. 2.2 Behavioral procedures

Four separate cohorts of mice were subjected to behavioral tests at 1 (1M) and 3 months (3M) after injury. One cohort of mice was subjected to cognitive tests, including Morris water maze and passive avoidance test. The second was subjected to the affective tests, including open field and shuttle box test. The third was subjected to homecage activity and cue-feared conditioning test. The fourth was subjected to social interaction test. The behavioral tests were separated by 5–7 days. The brain tissue was extracted for Western blot or sectioned for immunofluorescence analysis. The spleens were collected for the determination of T cell function and nuclear NF-κB translocation, and the plasma for stress hormones measurement. 2.3 Locomotor activity Homecage locomotion was performed in a floor area of 26×20 cm2 and recorded for a 10 min period at

Journal Pre-proof light (14:00 pm) and night (2:00 am). The spontaneous locomotor and exploratory activities were performed in open arena (50×50 cm2) (Xinruan Information Scientific Technology) for 30 min, from which the first 5 min was regarded as a measure of novelty-induced exploratory activity (Rosa et al., 2003). Total distance and velocity were determined from the video records using EthoVision 9.0 system (Noldus Information Technology, Netherland). The analysis of the behavior at the first 5 min in the open field test was performed by dividing the floor into 16 rectangles (4×4), in which the 4 central rectangles (2×2) was defined as the central zone. The distance travelled and the duration in the central zone is usually regarded as a measure of anxiety.

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2.4 Morris Water Maze (MWM) test

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MWM that assesses mouse spatial learning and memory was carried as reported (Acharjee et al., 2013;

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Anderson et al., 2015). Mice were first subjected to training trials for 5 consecutive days by sequentially placed in the each quadrant and allowed to search for the platform. On Day 6, mice were subjected to a 60

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s probe trial in which the platform was removed. In the following “reversal” phase of the MWM, the

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platform was reintroduced to the opposite quadrant of the pool, and mice were trained for consecutive 3 days in order to find the escape platform. On Day 10, the mice were subjected to the reversal probe trial in

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which the platform was removed again. The video was recorded by a digital camera and analyzed by the Noldus EthoVision 9.0 system (Noldus Information Technology) (Radjavi et al., 2014).

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2.5 Step-through passive avoidance test

The test includes two sessions: training and learning, with each 5 min performed in an instrument that contains two identical compartments (L×W×H in cm, 13.6×15.3×25), one in the bright and the other in the dark by a partition with a window (Wang et al., 2011). The floor of the dark compartment is composed of stainless steel rods connected to an electric shock generator. After 5 min’ habituation, once the animals stepped into the dark box, they received an electric shock (0.25 mA, 60Hz) delivered through the grid floor and lasted for 10 s. The electric shock was terminated when the animals crossed to the light box followed by a 10 s interval. One day later, the same procedure, but without footshock, was used for testing avoidance memory by measuring the latency, the entry, and the duration spent in the dark side. The video was recorded

and analyzed by DigBehav System (Jiliang Software Co., Shanghai, China). 2.6 Shuttle box active avoidance test The two-way active avoidance acquisition is performed in an automatic shuttle box (Xinruan

Journal Pre-proof Information Scientific Technology) with two identical boxes (L×W×H in cm, 13.6×15.3×25.0) connected by an opening (4×8 cm) (Lichtenberg et al., 2014). Each training and learning session consist 50 trials, except the former started after 5-min familiarization. Each trial consists of a conditioned stimuli (CS, light of 12W bulbs and sound of 2400 Hz at 40 dB, presented simultaneously, 20s) followed by an unconditioned stimulus (US, electric shock, 0.25 mA, 60 Hz, 10s) with 10 s interval. The stimuli were switched off as soon as the mice stepped into the other side of the shuttle box (Lopez-Aumatell et al., 2011; Lalanza et al., 2015). The active avoidance and passive escape are defined as the responses to CS and US respectively (Lopez-Aumatell et al., 2011). The movement were recorded and analyzed by DigBehav

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System (Jiliang Software Co.).

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2.7 Contextual and cued fear conditioning

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Contextual fear conditioning was used to assess the ability of mice to learn and recall an association between a novel environment (context) and a negative stimulus (footshock) (Ganon-Elazar and Akirav,

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2012; Harre et al., 2008; Kahn et al., 2012). The conditioning was performed in a square chamber

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apparatus (L×W×H in cm, 25×25×40) (Yuan et al., 2016). The training session consists of a 2 min exploration period as baseline and followed by 2 times of conditioned/unconditoned stimulus (CS-US)

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pairings (CS: tone 80 db white noise, 28 s duration; US: foot-shock intensity 0.2mA, 2s duration) separated by 1 min interval. Testing for freezing was performed 24 h later in the same training chamber for 5 min.

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Approximately 2 h after contextual testing, mice were placed into an altered context with same size, only the sound was given and lasted for 3 min. The movement was record by DigBehav System (Jiliang Software Co.) and analyzed by Noldus EthoVision 9.0 system. Freezing to the same context was used as a measure of contextual memory, whereas the freezing during the CS as a measure of cue-feared memory (Lin and Hsueh, 2014; Lotfipour et al., 2013). 2.8 Three-chambered social test Sociability was carried out in a three-chamber system with an open middle section for the mouse to enter or leave either chamber. Two identical wired cylinders were placed in each side of the chamber. In session I, a mouse (stranger 1) was placed under one of the wired cylinders, while the second wired cylinder was empty. In session II, a second (novel) mouse was placed under the cylinder that had been empty during session I. In each session, the test mouse was put into the middle chamber and was left moving freely for 10 min. The time spent by the subject mice in each chamber during each session was

Journal Pre-proof recorded. The stranger mice and the test mice were the same sex, weight, and age, but were not littermates of each other (Torres et al., 2016). 2.9 Forced swimming test This test measures depressive-like behavior with immobility taken as the dependent measure of behavioral despair (Weaver et al., 2005). Mice were individually forced to swim in a transparent open cylindrical container (diameter 10 cm, height 25 cm), containing 20 cm of water (depth) at 24 ± 1°C. A camera positioned at the same horizontal level of the cylinder recorded the 6 min swim session using the Noldus EthoVision 9.0 system. The inactive movement is described as behavior only necessary to keep its

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head above the water (Brocardo Pde et al., 2008; Kaster et al., 2005); the moderate activity is defined as

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only limbs movement, active activity as the moderate movement of four limbs and body, high activity as

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the quick movement of the whole body. 2.10 Sucrose preference

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Preference for a sucrose solution over water is regarded as a measure of anhedonia. Mice were

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individually caged with a free choice of tap water or a 1% sucrose solution for 2 consecutive days. Water and sucrose intake was measured daily and the position of the two bottles was switched every 12 h to avoid

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any side preference. Sucrose preference was calculated as [sucrose consumed/ (water consumed + sucrose consumed)] for each mouse (Anderson et al., 2015).

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2.11 Laboratory investigation

2.11. 1 Immunofluorescence analysis Brains sections were detected for BDNF (1:200, #SAB2108004, Sigma-Aldrich, Shanghai, China), ionized calcium binding adaptor protein 1 (Iba-1, 1:200; #ab108539, Abcam, MA, USA) and glial fibrillary acidic protein (GFAP, 1:200; #MAB360, Millipore, USA) by immunofluorescence as reported (Zhang et al., 2011). Images were obtained on a confocal laser scanning microscope (LSM 700, Carl Zeiss MicroImaging, Jena, Germany). 2.11.2 Western blot Nuclear protein from splenocytes was purified by Nuclear-Cytosol Extraction kit (Applygen Technologies Inc., Beijing, China). Brains were dissected into different regions and protein was extracted by RIPA solution. Brain or splenocyte samples were separated by PVDF gel and transferred onto polyvinylidene difluoride membranes (Millipore) followed by the appropriate primary antibodies for the

Journal Pre-proof brain [synaptophysin (#MAb5258, 1:1000, Millipore), BDNF (1:1000, Sigma-Aldrich), p-ERK (1:2000, #4370, Cell Signaling Technology), goat anti-mouse acetylcholinesterase (AchE, 1:100, #sc6430, Santa Cruz), Iba-1 (1:1000, Abcam), GFAP (1:1000, Millipore), anti-D1 dopamine receptor (D1DR, 1:200, #sc33660, Abcam), anti-D2DR (1:200, #sc5303, Abcam)] or the splenocyte [anti-NF-κB p65(ser) (1:1000, #3033,Cell Signaling) and NF-κB p65 (1:1000, #6956, Cell Signaling)]. After incubation with peroxidase-conjugated secondary antibodies (1:5000, Cell Signaling Technology), the signals were visualized by ECL reagent (Applygen Technologies Inc.) and exposed on ImageQuant LAS 4000 system (GE Healthcare, Germany). Membranes were stripped and reprobed with antibodies of ERK (1:2000,

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#9107, Cell Signaling) or β-actin (1:1000, Cell Signaling) for brain samples, and Lamin B1 (1:10000,

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#ab133741, Abcam) for splenocytes.

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2.11.3 T lymphocytes immunity

Splenocyte were cultured in complete RPMI-1640 medium in 96-well microtitre plates (4×105 cells per

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well) stimulated by concanavalin A (5 mg/L; Sigma) for 48h (Zhang et al., 2011). T cell proliferation was

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examined using a CCK-8 method (Dojindo Molecular Technologies Inc., Shanghai, China) in a multiplate spectrophotometer (Spectra MR; Dynex, Richfield, MN, USA) and IL-2 secretion by ELISA (ExCell

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Biology Inc., Shanghai, China).

2.11.4 Blood collection and plasma hormone analysis.

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The mice were anesthetized and the blood were drained from the suborbital vein and collected in tubes containing 5µl heparin. Plasma was separated by centrifugation (1,200 g×10min) at 4°C and immediately stored at -80°C until subjected to biochemistry examination. Stress hormones were measured by a competitive immunoassay (Jiancheng Life Sciences, Nanjing, China) following manufacturer’s protocol. 2.12 Statistical analysis Results are expressed as means ± SEM. Statistical analyses were performed using two-way ANOVA on the bodyweight, MWM training, step-through test, locomotion, light-dark transfer test, shuttle box test, and sucrose preference followed by Bonferroni’s or Tukey post-hoc comparisons. Two-tailed Student’s t test was used for the rest of the experimental results. The nonparametric data was analyzed by Mann-Whitney U test. P<0.05 was considered statistically significant.

3. Results

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General appearance of animals and weight loss following burn injury The wound on the back of burn mice healed at 2 weeks and the fur was recovered at 1 month postburn.

Weights displayed progressive loss from the 3 rd week till the 8th week. Two-way ANOVA analysis revealed a significant effect of burn injury (F(1,165)=4.528, P=0.035) and time (F(7,165)=4.138, P=0.001) on weight loss, without any interaction between the two factors (F(7,165)=0.927, P=0.465) (Fig.1A). 3.2 Burn injury promotes locomotion in homecage and open field tests To rule out the neurobehavioral changes induced by burn injury be possibly affected by motor function, we firstly observed their locomotion in homecage and open field tests. In the former, the burn mice

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displayed higher locomotion by traveling extremely longer distance with quicker speed than the sham mice

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during both light and night cycles (Fig.1B). Likewise, in the later, there were significant main effects of

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burn injury (1M: F(1,72)=32.798, P=0.000; 3M: F(1,138)=6.139, P=0.014) and time (1M:F(5,72)=4.949, P=0.001;3M: F(5,138)=10.561, P≤0.001) on the distance traveled without any interaction between the two

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factors (1M:P=0.902; 3M: P=0.219) (Fig.1Ci,V). Comparatively, the two groups differed significantly in

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the velocity (1M:F(1,72)=256.612, P≤0.001; 3M:F(1,138)=4.814, P=0.030) without any interaction between burn injury and the time (1M:P=0.763; 3M:P=0.051).Taken together, these results indicated that the higher

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locomotion be promoted absolutely by burn injury which persisted as long as 3 months postburn. A decrease in time spent in the central area of the arena is considered as an index of anxiety. In the

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open field test, burn injury exerted significant effect on the higher occurrence in the periphery of the arena (1M: F(1,72)=31.344, P≤0.001; 3M:F(1,138)=6.139, P=0.014), implying an anxiety-like behavior of burn mice (Fig.1C II, Vi). The above behavior was further corroborated by an increased propensity toward anxiety as with reduced ratio of center/total duration (Fig.1C IV, Viii) and distance (Fig.S1A) during the first 5 min in the open field. This preference was also represented by the path traces in Fig.S1B. 3.3 Burn injury improved the spatial learning and memory in MWM test Since cognition deficit is one of the typical behavioral phenotypes of PTSD patients, we firstly examined their spatial learning and memory by MWM test. In the 1M mice, during the acquisition phase of the MWM task (Fig.2A,upper lane), although there was a significant effect of training session on escape latency (F(4,160)=4.772, P=0.001), the learning curve did not differ significantly between the sham and burn mice in latency (F(1,160)=2.550, P=0.112), distance (F(1,160)=0.199, P=0.656), and the speed (F(1,160)=0.001, P=0.980), indicating an equivalent learning ability between the two groups. In the reversal training session,

Journal Pre-proof although the distance (F(2,96)=23.610, P≤0.001) and the speed (F(2,54)=12.799, P≤0.001) were simultaneously reduced with the training days in the two groups, there was no significant effect of training session on escape latency (F(2,96)=1.559, P=0.216), indicating that none of the two groups could relocate the platform effectively. Moreover, on the first day of reversal training, the burn mice took longer latency to reach the platform as opposed to the sham mice, implying relatively reduced flexibility in memory of burn mice. In the 3M mice, the effect of training session on escape latency was only close to its significance (F(4,170)=2.227, P=0.068), suggesting an attenuated learning ability of 3M mice relative to 1M mice.

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Although the learning curve did not differ significantly between the sham and burn mice in distance

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(F(1,170)=3.329, P=0.070) and the speed (F(1,170)=1.794, P=0.904), the burn mice took significantly shorter

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latency to reach the platform (F(1,170)=18.086, P≤0.001), indicating an improved learning ability in 3M burn mice. On the contrary, in the reversal training phase of 3M mice, the distance (F(2,170)=33.473,

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P≤0.001), latency to the platform (F(2,170)=19.525, P≤0.001) and the speed (F(2,170) =28.619, P≤0.001) were

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significant different within the training days, indicating the retained flexibility in memory of 3M mice. However, in comparison with the sham mice, burn mice from 3M group still took longer latency to

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relocate the platform (F(1,102)=11.021, P=0.001) and with lower velocity (F(1,102)=4.422, P=0.038) in the reversal training phase, indicating that burn injury could reduce the memory flexibility till 3M postburn.

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In the first probe trial of 1M mice (Fig.2A, lower lane), although the latency (P=0.069), distance (P=0.561), speed (P=0.411), and the entry in the target quadrant (P=0.306) were not different between the two groups, burn mice spent significantly more time (P=0.036) and traveled longer distance (P=0.034) in the target quadrant compared to the sham mice. Accordingly, the dwell time (P=0.034) and distance traveled (P=0.016) in the opposite quadrant were reduced simultaneously in burn as opposed to sham mice. However, in the reversal probe trial to examine the retention of memory, all the measures did not differ between the two groups, implying similar retention of the previous platform location after 3 days’ reversal training in 1M mice. Distinct from the 1M mice, 3M burn mice traveled longer distance (P=0.015) and with higher speed (P=0.015) than the sham mice in the first probe trial, however, the latency to reach the platform was comparable between the two groups (P=0.246). Similarly, burn injury prolonged the duration spent (P=0.053) and the distance traveled (P=0.055) in the target quadrant marginally, indicating the relative

Journal Pre-proof improved spatial memory lasting to 3 months postburn in burn mice. In the reversal probe trial, although the duration, latency, and distance in the target quadrant did not differ between the two groups, the burn mice still traveled relatively longer distance (P≤0.001) and with higher speed (P≤0.001) than the sham mice, suggesting the extremely high activity of burn mice. 3.4 Burn injury induced the passive avoidance behavior in step-through test To substantiate the observations in MWM and to rule out the stress related behavior, we next sought to assess other behavioral correlates of PTSD symptoms in a water-free step-through test (Fig.2B). Analysis of the two-day’s performance revealed a remarkable increased duration (F(1,63)=4.378, P=0.040) and

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distance (F(1,63)=10.957, P=0.002) in the light box of burn mice than the sham mice. In addition, the burn

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mice tended to complete fewer transitions as opposed to the sham mice (F(1,63)=3.023, P=0.087).

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Particularly on the learning session, burn mice took significantly prolonged latency to enter the dark box as compared to the sham mice (F(1,63)=9.253, P=0.003), indicating a typical passive avoidance behavior of

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burn mice. Moreover, there was a main effect of interaction of day×group on the latency (F(1,63) =16.875,

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P=0.0001.), distance in the light (F(1,63)=5.318, P=0.024.), transition (F(1,63)=5.601, P=0.021) and errors (F(1,63)=5.601, P=0.021), indicating the enhanced contextual fear learning and consolidation of burn mice

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relative to the sham mice.

However, in the 3M mice, there were neither significant difference in the percentage of duration

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(F(1,46)=0.417, P=0.522) and distance (F(1,46)=0.080, P=0.779) in the light box, nor in the latency into the dark box (F(1,46)=0.760, P=0.388) between the two groups. The disappearance of passive avoidance behavior in the 3M burn mice suggested a transient improvement in contextual fear learning and consolidation at 1M postburn (Kahn et al., 2012). 3.5 Burn injury attenuates the active coping ability in the shuttle box test For modeling behavioral phenotypes, avoidance of trauma-related cues is a critical feature of PTSD. With the above striking passive avoidance behavior of burn mice in hand, we next undertook the shuttle box test to study whether the active avoidance learning in response to auditory cue was also impaired in 1M burn mice. Analysis of the two days’ performance revealed that both sham and burn mice showed similar significant decline in the numbers of active avoidance (F(1,99)=14.207, P≤0.001) and passive escape (F(1,99)=17.144, P≤0.001) between the learning day and the training day, verifying comparable development of behavioral interference. Additionally, evaluation of latency to CS (F(1,99)=0.014, P=0.907)

Journal Pre-proof or US (F(1,99)=0.418, P=0.520) yielded no significant difference between the two groups. Nonetheless, as opposed to that on the training day, the sham mice rather than the burn mice displayed significant shorter immobile time in active avoidance response (Fig.3Ai) and more transition between the two boxes on the relearning day (Fig.3Aii). To make things worse, the burn mice tended to take longer time to cross the two boxes than the sham mice on the training day (P=0.087). Collectively, compared to sham mice, the burn mice showed deterioration in the active avoidance learning and consolidation of this task. 3.6 Burn injury enhances the contextual but impairs the cue-feared memory in the contextual fear conditioning test

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In order to further assess whether the cognitive differences between the two groups would be observed

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in a hippocampus-dependent task, we utilized a commonly used contextual fear task in which higher

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freezing time implying a strong association between the conditioned and unconditioned stimulus. As anticipated, no significant differences in freezing time existed between the two groups on the training day

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(P≥0.05), once again suggesting no sickness behaviors of burn mice after 1 month of injury. Unexpectedly,

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on the testing day, both sham and burn mice showed significant less freezing time to the 2 min contextual fear conditioning (CS, context A) than the control mice (Fig.3B). Specifically, burn mice showed

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significantly longer freezing time in response to CS than the sham mice (Fig.3B). On the contrary, burn mice other than the sham mice showed less freezing time to the 3 min auditory cue conditioning (context B)

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as opposed to the normal mice (Fig.3B). These data suggested that the contextual fear memory was improved while the auditory fear memory was impaired in burn relative to sham mice. 3.7 Burn injury induces depressive-like behaviors in social interaction and shuttle box test Sociability and social novelty preference tests detect changes in social withdrawal, modeling the depressive-like behavior of PTSD. We first performed the sociability test using the test equipment containing an empty wire cage and a wire cage apparatus keeping a stranger mouse (Stranger 1). Multiple factors ANOVA analysis revealed significant longer time for control and sham mice spent in the chamber with the cylinder containing mouse than in the chamber with empty cylinder for session 1 (P<0.01). By contrast, burn-injured animals did not display a preference for either chamber (Fig.3Ci, P>0.05). Following up with specific novel mouse comparisons in session 2 revealed that both control and sham mice spent more time with the cylinder containing a novel conspecific mouse (stranger mouse 2) than a familiar one (stranger mouse 1) (Fig.3Cii, P<0.01). Once again, burn mice spent a similar amount of time

Journal Pre-proof in both chambers, indicating equal exploration of the maze. Together these results suggest a role for burn injury in social behavior inhibition. The deficit in active avoidance learning in the shuttle box test of burn mice may also be a sign of depression (Guo et al., 2013). Therefore, the performance on a depression-associated behavior was further examined in the forced swimming test. Paradoxically, the immobility time was reliably shorter in the burn mice than in the sham mice at both 1M and 3M groups. Strikingly, burn mice swam extremely longer distance with higher velocity compared to sham mice (Table). The anti-depressive behavior of burn mice in forced swimming test was further corroborated by the sucrose preference test, in which there was no

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significant difference between the two groups (F(1,68)=0.752, P=0.389) and no interaction between

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day×group on sucrose preference (F(1,68)=0.285, P=0.595) (Fig.4).

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3.8 Burn injury induced increased BDNF expression in the brain and Th2 switch in splenic T cells Since the cognitive behavior is a hippocampus-dependent task, therefore, the phenotype analysis of

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hippocampus and frontal cortex was performed by Western blot and immunofluorescence approaches.

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Microgliosis often occurred as a consequence of brain injury and a marker of neuroinflammation. To our surprise, long-lasting upregulation of Iba-1 (a marker of micrglia) was observed in the hippocampus

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(P≤0.001) (Fig.5A, C) but not frontal cortex (Fig.5B) of burn vs. sham animals from 1M and 3M mice. A trend of increased expression of GFAP (a maker of astrocyte) in the hippocampus from burn vs. sham mice

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was also found (Fig.5A). Moreover, BDNF expression was significantly upregulated in the CA2 area of hippocampus in burn-injured 3M mice and an increased trend in 1M burn mice compared to corresponding sham mice (Fig.5A, C). To quantify the findings, a significant induction in BDNF in both hippocampus (P=0.022) (Fig.5Aii) and prefrontal cortex (P=0.029) (Fig.5Bii) was observed in burn vs. sham mice by immunoblot (Fig.5Ai, Bi). The expression of AchE in the prefrontal cortex was also remarkably increased in 3M burn mice relative to the 3M sham mice (P=0.037). By contrast, the expression of synaptophysin, a pre-synaptic molecule, was decreased in the prefrontal cortex of burn vs. sham mice (P=0.046) (Fig.5Bii). In 1M mice, although D1DR expression did not differ between the groups, D2DR was significantly increased in the striatum of burn vs. sham mice (Fig.S2). New paradigm has been proposed involving simultaneous and rapid induction of innate genes (both pro- and anti-inflammatory) and suppression of adaptive immunity genes in trauma (Xiao et al., 2011). The long-lasting neuroinflammation in burn mice prompted us to detect the chronic splenic T cell

Journal Pre-proof immunity from both groups of mice. With respect to sham mice, in terms of proliferation and IL-2 secretion, T cell function was not significantly changed in burn mice at 1M postburn, except that T cells secreted increased IL-4 and IFNγ. With the time, the splenocyte number was significantly reduced in burn mice as opposed to sham mice at 3M postburn and with lower IL-2 secretion. Moreover, a predominant Th2 response with higher IL-4 but lower IFNγ secretions was found in the burn relative to the sham mice at 3M postburn (Fig.6). Accordingly, splenic nuclear NF-κB translocation was mildly increased in burn compared to sham mice, indicating sustained low degree of inflammatory response in 3M burn mice (Fig.S3) even the circulating proinflammatory mediators were not changed (data not shown).

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In accordance, none of the blood profile was aberrant at 3 months following burn injury when all the

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behavior procedures were completed (Table S1). The increased stress hormones at 2 h following burn

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injury were declined to the comparable level as the sham mice at 24 h, and remained at the baseline till 3

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months (Fig.S4).

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4. Discussion

This investigation demonstrated a long-lasting neuropsychological disorder in thermal-injured mice

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resembling human PTSD. These abnormalities include the heightened context fear memory in context conditioning test (Cluster B), heightened passive avoidance behavior in step-through test (Cluster C),

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worsened active avoidance capability in the shuttle box test (Cluster D), anxiety-like behavior in open-field test (Cluster D), deficit in preference for social novelty in social interaction test (Cluster D), hypervigilance in the homecage, open-field and forced swimming tests (Cluster E). In conflict, the burn mice showed improved spatial memory in Morris Water Maze test, no sign of depression in the forced swimming and sucrose preference tests. The neuropsychological disorder was associated with a concomitant microgliosis and significant increased hippocampal BDNF expression in the burn relative to sham mice. A switch toward Th2 response and increased NF-κB translocation were found in the splenocytes from the burn as compared with the sham mice. This psychological disorder is quite special because it occurs in the burn mice as long as 3 months after burn injury. Although the psychological impact of burn injury is widely recognized, an increasing number of studies have examined not only the prevalence of psychological difficulties of inpatients, but also the impact these alterations have on recovery (Wisely et al., 2007). In our study, the neurobehavioral tests

Journal Pre-proof were not performed until 1 month after burn injury to avoid the influence of unhealed wound. This interval is equivalent to about 3–3.5 years in the human according to the expected lifespan (Wuri et al., 2011). Therefore, our findings could actually be extended to humans in the recovery period of burn injury. 4.1 Burn mice showed improved spatial memory in MWM test Unexpectedly, burn mice in our observation obtained improved spatial memory with better orientation of the target quadrant and relative shorter latency reaching the platform in the MWM. This finding was somewhat unexpected, as in virtually every other study, burn injury was always associated with impaired cognitive performance in humans (Guo et al., 2017; Purohit et al., 2014) and lower discrimination memory

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in rats(Halm et al., 2006). This apparent discrepancy may be explained by a maladaptive behavior to

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unconditioned stimulus (e.g., thermal injury) in a safe environment (e.g., water) for the burn mice. It was

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suggested that a fear conditioning may become maladaptive when this adaptive behavior to an unconditioned stimulus in a dangerous environment becomes associated with another previously neutral

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stimulus in a safe environment (Ross et al., 2017). Therefore, the conditioned stimuli consistently elicit

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traumatic memories and flashbacks as in PTSD (Charney et al., 1993). In the reversal training phase of MWM, 1M burn mice took relative longer time to the platform on the first day, whereas the 3M burn mice

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took consistently longer time to the platform throughout the reversal training days. These findings suggested that similar to the deficits in extinction retention in single prolonged stress model of PTSD

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(Knox et al., 2012), burn mice have increased retention of the previous spatial memory, or in other words, were deficit in extinction of the spatial memory, mimicking the flashback of the trauma in PTSD patients. An increase in BDNF expression while a deficit in synaptophysin expression in the brain of burn relative to sham mice may account for these alterations. Alternatively, the improved cognition in MWM could also be explained by the ‘post-traumatic growth’, which indicates that the patients’ recovery exceeds pre-trauma levels of well-being (Baillie et al., 2014). There were also positive changes that emerge from distress and correlated with affective emotion in burn patients (Wei et al., 2017) and in animals with partial hepatectomy (Wuri et al., 2011) or heat stress preconditioning (Su et al., 2009) for unknown reasons. 4.2 Burn mice showed re-experiencing and heightened avoidance symptoms with impaired extinction of fear response To rule out the interference of the water in MWM test, we carried out the step-through passive

Journal Pre-proof avoidance test to examine the contextual fear memory of burn mice. Once again, compared to the sham mice, the burn mice showed heightened fear memory by spending significant longer latency into the dark box on the learning trial. These findings suggested that burn mice have increased retention of the previous contextual fear memory, or in other words, were deficit in extinction of the contextual fear memory, mimicking the flashback of the trauma in PTSD patients. The above finding prompted us to further examine their fear responses in fear conditioning and extinction test, which have been extensively used to study the excessive fear responses like PTSD by measuring the time spent freezing (Hill and Martinowich, 2016). Consistently, the burn mice showed

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longer freezing time as opposed to the sham mice in contextual fear expression, suggesting a deficit in

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contextual fear extinction. However, compared to the normal mice, burn mice other than the sham mice

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manifested a deficit in cue fear memory that with less freezing time to auditory stimulus on the fear expression trial. This distinct performance between context- and cue-based fear memories of burn mice

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was further corroborated in shuttle box test, in which deterioration in active avoidance learning and

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consolidation was found in burn vs. sham mice. Furthermore, burn mice took longer latency and stayed longer immobile time in active avoidance, indicating impaired stress-coping efficacy (Cigarroa et al.,

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2016). The coping efficacy that effectively deal with posttrauma recovery demands, its deficit indicates initial PTSD symptoms (Bosmans et al., 2015).

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The above mentioned discrepancy between contextual and auditory fear memory of burn mice was absolutely unusual, since the burn mice showed consistently cognitive improvement in both MWM and passive avoidance tests. It could be explained by the observations that contextual fear conditioning and auditory-cue fear conditioning depend on different neural processes (Pugh et al., 1999). In socially isolated rats, contextual fear memory was impaired while auditory-cue fear conditioning was intact; while only the former impairment could be prevented by IL-1 receptor antagonist (Pugh et al., 1999). This hypothesis was also reinforced by another study that administration of acyl-ghrelin could significantly impair long-term auditory fear memory without affecting contextual fear memory (Harmatz et al., 2017). The underlying mechanism deserves further study. 4.3 Burn mice showed a hypervigilance behavior but with negative mood Consistent with the hypervigilance behavior in the animal models of PTSD, burn mice showed profound increased locomotion than the shams in homecage and open field tests, as well as reliably longer

Journal Pre-proof mobility time in the forced swimming test. These intriguing hyper-arousal behavior in response to novel stresses (i.e., open field, constricted water) constitutes the main syndromes for PTSD, a disorder where learned fear due to a traumatic event becomes generalized to safe situations (Mahan and Ressler, 2012). Even so, as opposed to the sham mice, burn mice consistently exhibited an anxiety-like behavior with preference to the peripheral arena in the open field test. In accordance, the burn mice showed no sign of depression behavior, possibly due to the moderate %TBSA exposure in our animal model. To examine the effect of burn injury on sociability that constituting a crucial component of mood behavior, we performed social interaction tests in 1M mice. Overall, social interaction with a novel

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environment revealed a significant inhibitory effect of burn injury on the preference of chamber containing

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the mouse to an empty cage, or the strange mouse to a familiar one. The deficit in sociability and social

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novelty preference recapitulated the above mentioned negative mood resembling PTSD. To rule out the interference of hyperlocomotion of the burn-injured mice, we presented the results with the total duration

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other than the distance traveled in each chamber.

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4.4 Neurobiological mechanisms for PTSD in burn mice

To understand and better treat fear-related disorders, identifying the processes occurring during

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association of contextual and sensory cues with trauma is also critical. There are numerous plausible mechanisms by which the burn mice might experience improved cognition even though moderately

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irritated. First, the increased expressions of BDNF that are involved in the memory extinction may account for the improved spatial memory or the heightened fear memory of burn mice. BDNF signaling appears to play a significant role in synaptic plasticity underlying the consolidation and the persistence of fear memories. The upregulation of BDNF in the brain of burn vs. sham mice was intriguing. In many models of trauma or neurodegenerative diseases, BDNF expressions were reduced, resulting in declined cognition (Wang et al., 2017). Except that in the models of traumatic brain injury (TBI) (Madathil et al., 2017), orthopedic surgery (Zhang et al., 2016), and stroke(Jiang et al., 2016), a brief glial BDNF was transiently upregulated in both cortex and hippocampus and even invaded the ischemic core (Jiang et al., 2016). In these cases, the glial production of BDNF may contribute to the increased expression of BNDF (Parkhurst et al., 2013). It was reported that microglia/macrophage undergo prolonged activation after stroke or TBI, and express BDNF and its receptors in the primate brain to induce endogenous neurogenesis against ischemic brain injury and the repair following TBI (Song et al., 2016). In addition, the glial production of

Journal Pre-proof BDNF played a crucial role in the therapeutic effect of antidepressants (Hisaoka-Nakashima et al., 2016). We suppose that the increased cerebral BDNF expression in burn injury could possibly serve as a compensate mechanism for brain injury as a result of the persistent activation of microglia as long as 3M postburn. On the other hand, the upregulation of BDNF in the brain of burn mice could also result in worsened flexibility in the reversal training of MWM, which may also be attributed by the reduced expression of synaptophysin in burn mice. Secondly, increasing studies suggested that impairments in cognitive and affective behaviors may be modulated, in part, by T cell homeostasis (Kipnis et al., 2012). Brain antigen-reactive CD4+ T cells are

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sufficient to support learning behavior (Radjavi et al., 2014; Wolf et al., 2009) by secreting IL-4 to

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regulate an anti-inflammatory phenotype in meningeal myeloid cell (Derecki et al., 2010) and BDNF

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expression (Derecki et al., 2010; Ziv et al., 2006). By contrast, the mice lacking T cell receptor showed reduced anxiety-like behavior compared to wild type (Rilett et al., 2015). In particular, CNS-specific shift

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toward Th2 phenotype at the choroid plexus was affected by peripheral immunosenescence in aging brain

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(Baruch et al., 2013). Similar to the findings of a Th2 shift following burn injury (Guo et al., 2003; Zang et al., 2004), we found that burn injury elicited a Th2-type response as long as 3 month after injury. In

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accordance with the finding that the burn-induced Th2 response resulted in a decreased resistance to subsequent infection (Guo et al., 2003; Zang et al., 2004), 3M burn mice showed relative reduced number

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of splenocyte but increased nuclear NF-κB translocation into the splenocyte, suggesting a chronic low-degree pro-inflammatory status.

Thirdly, the increased expression of BDNF may also account for the hyperarousal status of burn mice. The impulsiveness observed in patients with high levels of hyperarousal was found to be associated with greater BDNF production (Martinotti et al., 2015). The increased BDNF production in the brain of burn mice could result in the deficit in fear extinction, therefore, a hyperarousal behavior was evoked in the irritated burn mice. The burn-induced hypervigilance could also be explained by the neurobiological findings of the increased expression of D2DR in the striatum from burn vs. sham mice. It was reported that altered striatum dopamine transporter and D2DR, but not D1DR densities were found in the striatum of an animal model of PTSD (Enman et al., 2015). Moreover, dopamine-dependent increased basal locomotion either in the striatum or nucleus accumben was caused, at least in part, by elevated D2DR expression (Dadalko et al., 2015; Gallo et al., 2015). It could be possibly that the burn-induced elevated D2DR

Journal Pre-proof expression be associated with the high hypervigilance of burn mice.

5. Conclusions Our findings are of considerable interest since they indicate a long-lasting neuropsychological impairment that resembling human PTSD in the aftermath of burn exposure. Although the precise mechanism by which burn injury induced behavioral and neurobiological modifications remains to be clarified, our findings suggest a crucial contribution of neuroinflammation as a consequence of prolonged Th2 type response in the etiology. Furthermore, the results extended previous findings in acute model of

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PTSD to a chronic post-trauma neuropsychological consequence in burn injury that is more relevant to

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clinical practice. Our study adds to the growing body of data pointing to an adaptive immune therapeutic

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potential of the psychological problems.

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Author Contributions:

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QHZ: the conception, formulation and design of the study, supervision the experiments, interpretation all the data, drafting and final approval the paper. JWH: mainly conduction the experiments and

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acquisition all the data. XJJ: examined T cell function. GLL: conduction the homecage and social interaction tests. MZ: critical review and comment on the manuscript. YMY: Acquisition part of the

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financial support and provided the personnel.

Conflict of Interest None

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Figure legends: Fig.1 Burn injury promoted the spontaneous and locomotor activities at 1 month (1M) and 3 months (3M)

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following injury. (A) Body weight was monitored over the whole period of experiment. n=10-20/group. (B)

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Spontaneous activity in homecage was monitored at day and night cycles for 10 min each. n=6-8/group. (C) Locomotive and exploratory activity in the open field. Total distance traveled, peripheral frequency, and

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center/total duration in 5-min bins over a 30-min test period was recorded by the Noldus system. The

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exploratory activity was represented as the center/total duration at the first 5 min in open field. Burn mice exhibited an anxiety-like behavior by showing reduced ratio of the duration in the center field compared

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with sham mice. Values are Mean ± SEM of sham vs. burn mice in 1M group (n=19 vs.17) and 3M group

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(n=11 vs.14) mice. #P<0.05, ##P<0.01 vs. normal mice, *P<0.05, *P<0.01, ***P<0.001 vs. sham mice by

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two-way ANOVA followed by Tukey post-hoc comparison or Student t-test.

Fig.2 Burn injury improved the spatial memory in Morris water maze (A) and contextual fear memory in step-through tests (B) at 1 month (1M) and 3 months (3M) following injury. (A) Spatial learning performances and cognitive flexibility including a 5-day’ acquisition phase and a 3-day’ reversal phase, followed by a probe session without the platform each (upper lane). Short-term memory and cognitive flexibility were assessed during the probe test (probe1) on day 6 and the reversal probe trial (probe 2) on day 10, respectively. Latency to reach the platform and the dwell time in the quadrants were recorded (lower lane). (B) The contextual fear memory was assessed by the passive avoidance test. On the learning trial, the burn group took longer latency into the dark compartment compared with the sham group or itself the day before. However, the improvement was absent in sham group. Values are Mean ± SEM of 17~25/group. *P < 0.05, **P < 0.01, ***P<0.001 vs. sham group, #P<0.05, ##P<0.01 vs. the training day, as analyzed by two-way ANOVA followed by Tukey post-hoc comparison or Student t-test.

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Fig.3 Burn injury impairs the cued-fear memory consolidation and social interaction in mice. (A) The consolidation of a conditioned avoidance to tone (active avoidance) and unconditioned avoidance to electric shock (passive escape) in burn vs. sham control in the shuttle box. The development of behavioral interference is evident in sham rather than the burn mice that display significantly less immobile time to tone and more transition between the two boxes during the learning compared with that in the training session (2-way ANOVA, P<0.05). n=29/group. *P<0.05 vs. sham. (B) Context (context A) and auditory associative (context B) memory as shown by the freezing behavior in the fear conditioning test. The former

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was enhanced while the latter was impaired in the burn vs. sham mice. n=5-20. *P<0.05, **P<0.01 vs.

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control; #P<0.01 vs. sham. (C) Burn mice showed deficit in sociability (i) and preference of novel mouse

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(ii) than the control or sham mice. n=15/group. The values represent Mean ±SEM of 1M mice. **P<0.01

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vs. control by Mann- Whitney U test or Student t-test.

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Fig.4 Burn injury augmented neuroinflammation and neurotropic factor in the brain of mice. (A, B) Immunoblots (Ai, Bi) and densitometric analyses (Aii, Bii) of synaptophysin (Syn), p-ERK,

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acetylcholinesterase (AChE), microgliosis marker Iba-1, astrocyte marker (GFAP), and neurotrophin BDNF in the brain from hippocampus (A) and frontal cortex (B) of mice 1 month (1M) and 3 months (3M)

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after injury (n=4-6 per group). (C) Fluorescence images of microglia activation markers in the cortex and BDNF in the hippocampus of 3M mice. *P<0 .05 vs. sham mice.

Fig.5 Long-lasting shift to Th2 response of splenic T helper cells following burn injury. Splenocytes harvested from sham or burn mice 1 month (A) or 3 months postburn (B) were cultured in the presence of concanavalin A (5 mg/L) for 48 h. T-cell viability was determined by CCK-8 method and the supernatants were collected for cytokine analysis. Data were shown as mean ± SD for n=8/group from 2-independent tests. *P<0.05, **P<0.01, ***P<0.001 vs. sham mice by Student t-test.

Table Effects of burn injury on the activity of mice in the forced swim test which were recorded at 5 s intervals throughout the 5-min test period. Data represent Mean ±SEM of sham vs. burn in 1M (n=11 vs. 9) and 3M (n=11 vs.14) groups.

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Supplemental materials Fig.S1 Locomotive activity and representative path traces in the first 5 min in the open field as recorded by the Noldus system. (A) Burn mice 3 months postburn exhibited an anxiety-like behavior by showing reduced ratio of the distance in the center field compared with sham mice. (B) Inner box represents the center zone. Data are presented as Means± SEM. n=10 per group. ***P<0.001 vs. sham mice.

Fig.S2 Burn injury did not induce depression-like behavior in sucrose preference test. Sucrose preference

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was expressed as the percentage of 1% sucrose solution intake for two consecutive days in sham vs. burn at

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3 weeks (n=11 vs. 10), 6 weeks (n=11 vs.14) and 3 months (n=11 vs. 14) post injury. Data are presented as

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Mean ± SEM. No significant difference was found between the sham and burn mice at different time points as analyzed by two-way ANOVA, except that the burn mice drank less sucrose compared to sham

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mice on day 1 at 6 weeks pb. *P<0.05 vs. sham mice of the same day by Mann-Whitney U test.

Fig.S3 Elevated expression of D2DR but not D1DR in the striatum of 1M burn vs. sham mice. β-actin was

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probed as a loading control (n =4).

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Fig.S4 Burn injury induced moderate nuclear NF-κB translocation into splenocyte at 3 months postburn. Representative immunoblots of p65-NF-κB and NF-κB expressions in the splenocyte of burn and sham animals. Lamin B1 was probed as a loading control.

Fig.S5 Burn injury transiently induced peripheral hormones levels in mice. Effect of thermal injury on serum norepinephrine (A), epinephrine (B), and corticosterone (C) in Balb/c mice after 2h, 24h, and 1, 2 or 3 months (M) post injury. n=8-12 per group. *P<0.01 vs. sham mice by Student t-test.

Table S1 Serum profile in 3M burn mice. The results are expressed in Mean ± SEM of n=11 vs.11 per group. **P<0.01 vs. sham mice. AST: Aspartate transaminase, ALT: Alanine aminotransferase, HDL: high density lipoprotein, LDL: low density lipoprotein, VLDL: very low density lipoprotein

Journal Pre-proof Table 1. Effects of burn injury on the activity of mice in the forced swim test.

Cumulative Duration (s) Grou p (pb)

ment

Moderately

Inactive

Activ

active

e

185.85± sham 1 month

Distanc

Treat active

35.56

15.30

76.68±7.49

±3.83

114.98± burn

Highly

56.77

21.07

87.52±9.33

±5.30

e moved

city

(cm)

(cm/s)

34.83±

572.65±

4.33

35.42

63.52±

736.04±

7.86

66.82

0.012

0.371

0.004

0.004

0.035

39.14

45.80±

637.09±

4.93

38.01

72.65±

859.25±

±3.88

6.20

32.73

of

value

136.19±

3 burn

15.31

77.60±6.65

58.23±1

111.47±6.0

1.77

±4.33

63.21

-p

sham

9

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P 0.001

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0.000

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value

1.56 ±0.10 2.01 ±0.18 0.03

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P

months

Velo

0.000

5 1.88 ±0.10 2.39 ±0.09 0.00

0.004

0.000

1

Journal Pre-proof Highlights 

Burn mice manifested long-lasting improved spatial and contextual fear memory and

increased

locomotion. However, the cue fear memory and consolidation was impaired in burn mice, along with worsened coping ability and loss of interaction in sociability. The above behavioral alterations corresponded to the re-experiencing, avoidance, arousal, and negative mood symptoms in PTSD. 

Burn mice showed sustained increased microgliosis and astrocytosis along with increased BDNF expression in brain.

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The long-lasting shift to Th2 response in burn mice may attribute to the neurobehavioral alterations.

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

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