Blast-induced traumatic brain injury (bTBI) is a significant medical and health concern in trauma surgery, both during peacetime and in wartime1. More than 20% of people working in explosion-related industries suffered a closed head injury as a result of blast overpressure (BOP) exposure, resulting in headaches, insomnia, anxiety, depression, memory and disorientation, placing a heavy burden on their families and society2,3. Due to the mild brain injury symptoms being not obvious, it is easy to be missed or misdiagnosed, thus timely identification and proper managing of blast-induced brain trauma are critical since traumatic brain injuries (TBIs) often worsen in clinical outcome within 48 h if not identified timely or not treated appropriately4.

Clinical reports have indicated the development of cognitive associated disorders following BOP exposure. The majority of these disorders are associated with anxiety, attention deficits, memory issues and impaired /altered problem solving skills3. Overlapping symptoms with other forms of trauma, such as non-blast related traumatic brain injury (TBI) and post-traumatic stress disorder (PTSD) have confounding effects on differential diagnosis. Animal models of shock wave injuries have been established to investigate injury mechanisms from overpressure exposure and its subsequent neurological impairments. Most published studies are limited to a single overpressure exposure or repeated exposures at the same magnitude of overpressure5,6,7,8,9,10. Researchs11,12 employed a rodent model of primary blast neurotrauma to determine the pressure at which acute neurological alterations occurred, while information on the neurological behavior and serum molecular changes in response to varied levels of overpressure at chronic stage is still unknown.

The role of neuroprotective cascades varied with different overpressure magnitudes. The sustained increase in the cascade of cell damage and death requires neuroprotection in order to begin neuroplasticity and regeneration. BDNF is known neuroprotective protein which is released after injury to facilitate repair mechanisms13 and also contribute to functional recovery after TBI. Neurotransmitter theory is the main theory of neurobiological pathogenesis of anxiety disorder. The transmission of information in the nervous system is transmitted through neurotransmitters, which are divided into monoamines, amino acids and so on.

In this study, a comprehensive evaluation of neurological function including learning, memory and anxiety-like behavior were performed utilizing Multi-Conditioning System (MCS). Enzyme-linked immunosorbent assay (ELISA) will be employed to elucidate protein changes associated with neural function, while protein sequencing will be used to identify serum biomarkers that can rapidly diagnose and predict brain injury to make judgments and interventions in the shortest time after shock wave exposure. By combining these approaches, we seek to identify overpressure thresholds that elicit anxiety-like behavior and memory impairment and to characterize the pattern of temporal changes following injury. This research could potentially reveal novel targets for drug development and efficacy monitoring in bTBI.

Results

Shock Wave Overpressure simulated exposure

After subtracting the baseline value, the three types of diaphragm rupture generated shock waves with the following overpressures as measured by the sensor: (65.3 ± 1.5) kPa, (91.7 ± 1.3) kPa, and (120.3 ± 10.5) kPa. The duration of the positive pressure was (4.4 ± 0.3) ms, (4.9 ± 0.7) ms and (6.3 ± 1.3) ms, respectively (Fig. 1).

Fig. 1
figure 1

The time-pressure profile of a shockwave recorded near the head of the rat. Schematic diagram of biological shock tube(A). 1. High pressure gas; 2. High voltage drive section; 3. Diaphragm; 4. Low voltage driven section; 5. Rat observation window; 6. Overpressure sensor. The arrow pointed in the direction of shock wave propagation. Shock wave simulated exposure in rats (B). Representative pressure profiles of low (60 kPa, C), moderate (90 kPa, D) and high (120 kPa, E) shock wave overpressure.

Full size image

Moderate and High Levels Shock Wave Overpressure Exposure Rats Displayed Anxiety-Like Behavior at 1d post blast

Locomotor activity in light–dark box test and open field test was assessed to evaluate the anxiety-like behavior of rats. At 7 d post-blast, no significant differences were found in light–dark box test or open field test between the Sham group and the blast-injured groups (60 kPa, 90 kPa, and 120 kPa). At 1 d, the 90 kPa and 120 kPa groups showed a significant decrease in number of visits and time spent in light in the light–dark box test compared to the Sham group (Fig. 2A, B and E, one-way ANOVA, F (3, 40) = 8.67, F (3, 36) = 8.39, p < 0.05). Similarly, distance traveled and number of visits in center in open field test were also comparable between the two groups (Fig. 2C, D and F, one-way ANOVA, F (3, 36) = 5.66, F (3, 36) = 4.04, p < 0.05). At 28 d, the 120 kPa groups exhibited significantly decreased distance traveled and number of visits in center in open field test compared to the Sham group (Fig. 2C and D, p < 0.05). At 90 d, the 120 kPa groups exhibited significantly decreased all parameters compared to the Sham group (p < 0.05). Track plots showed that the 120 kPa group moved less in the light box and the center of open field than the Sham group at 1 d and 90 d after exposure (Fig. 2E and F). These findings suggest that exposure to moderate and high intensities overpressure (90 kPa and 120 kPa), may induce anxiety-like behaviors in rats at an early stage. Over time, anxiety-like behaviors in the medium-intensity group were relieved, while behavioral abnormalities in the high-intensity re-emerged.

Fig. 2
figure 2

Light-dark box and oped field experiment outcomes following Blast Exposure. Number of visits in light (A) and time spent in light (B) of rats in the light–dark box examination. Distance traveled in center (C) and number of visits in center (D) in open field examination. (n = 12 per group) (*p < 0.05 vs. Sham, &p < 0.05 vs. 60 kPa). Trajectory of rats in light-dark box test (E). Left compartment is light box, right compartment is dark box. Trajectory of rats in open field test (F).

Full size image

High Level Shock Wave Overpressure Exposure Rats Displayed inferior learning and memory at 1d post blast.

Nondeclarative memory, which involves learning and retaining skills and habits without conscious recall, was assessed using an active avoidance training paradigm. At 1 d after exposure, interaction between number of active avoidance group and time, main group effect and main time effect were all significant (Fig. 3A, repeated measures ANOVA, F (9, 108) = 3.79, F (2.6, 94.3) = 239.40, F (3, 36) = 20.65, all p < 0.05). It is suggested that with the increase of training time, the ability of rats to avoid harmful stimuli in each group has a tendency to improve, but the improvement of the ability in all exposure groups is lower than Sham group. The simple effects within time analysis showed rats in the 120 kPa group had a significantly fewer times on the last training trial (p < 0.05), suggesting an impairment of memory formation. Interaction between active avoidance reaction time group and time was not significant (F (9, 108) = 0.08, p > 0.05). A probe test conducted on the 6th day further revealed a significant reduction in active avoidance times in the 120 kPa group compared to the Sham group (Fig. 3C, one-way ANOVA, F (3, 32) = 9.35, p < 0.05), indicating insufficient long-term memory storage after extensive training. No significant differences in active avoidance times (Fig. 3E, G, I, K) and reaction time (Fig. 3B, D, F, H, J, L) were observed among the groups during other training trials or probe tests. These findings suggest that shock wave overpressure exposure, particularly at the 120 kPa level, may selectively impair the formation and consolidation of nondeclarative memories in rats.

Fig. 3
figure 3

Active avoidance experiment outcomes following Blast Exposure. Active avoidance times (A) and reaction time (B) in training days from 2 d to 5 d post blast. (C) and (D) in probe test at 6 d post blast. (E) and (F) in training days from 29 d to 32 d post blast. (G) and (H) in probe test at 33 d post blast. (I) and (J) in training days from 91 d to 94 d post blast. (K) and (L) in probe test at 95 d post blast. (n = 12 per group) (*p < 0.05 vs. Sham).

Full size image

Moderate and high levels shock Wave Overpressure reduces serum levels of biomarkers

To investigate the effects of shock wave overpressure exposure on neurotransmitter levels, we measured serum levels of DA, 5-HT, BDNF and GABA at 1 d, 7 d, 28 d, and 90 d post-exposure. Both the 90 kPa and 120 kPa groups displayed significantly lower DA levels compared to the Sham group at 1 and 28 days (p < 0.05) (Fig. 4A). This decrease in DA, a neurotransmitter crucial for reward, motivation, and motor control, may underlie some of the observed behavioral deficits. 120 kPa group exhibited significantly reduced 5-HT levels at 1 and 90 days (p < 0.05) (Fig. 4B). This finding suggests potential disruption in serotonergic pathways, which are implicated in mood regulation and anxiety. The 60 kPa group exhibited elevated BDNF levels compared to the Sham group at 7 and 28 days (p < 0.05) (Fig. 4C), suggesting a potential compensatory response to the shock wave overpressure. Conversely, the 120 kPa group showed significantly lower BDNF levels than the Sham group at 90 days (p < 0.05), indicating a long-term impact on neurotrophic support. No significant differences in GABA levels were observed between the blast-exposed groups and the Sham group at any time point (Fig. 4D). This observed alterations in neurotransmitter levels provide further evidence for the neurobiological effects of shock wave overpressure exposure, potentially contributing to the observed behavioral changes.

Fig. 4
figure 4

Changes of DA, 5-HT, BDNF and GABA protein levels in serum at 1 d, 7 d, 28 d and 90 d after explosion (n = 5 per group) (*p < 0.05 vs. Sham, &p < 0.05 vs. 60 kPa, #p < 0.05 vs. 90 kPa).

Full size image

Screening and confirmation of differentially expressed proteins(DEPs)

To identify proteins potentially impacted by shock wave overpressure exposure, we performed a differential proteomics analysis using a strict threshold of unique peptide ≥ 1, fold change ≥ 1.5 or ≤ 0.67, p-value or p-value-chi-test < 0.05, and FDR < 0.05. Among the DEPs, 162 proteins were identified as significantly differentially expressed between the sham and the 60 kPa group (p < 0.05), including 76 upregulated and 86 down-regulated proteins. 533 proteins were identified as significantly differentially expressed between the sham and the 90 kPa group (p < 0.05), including 197 upregulated and 336 downregulated proteins. 220 proteins were identified as significantly differentially expressed between the sham and the 120 kPa group (p < 0.05), including 58 upregulated and 162 down-regulated proteins (Fig. 5A). Visual analysis results of differential proteins showed that upregulated proteins include Rabif, aldoa, Pepd, etc. and downregulated proteins include Gip, Mrm3, Hp, etc. in the 60 kPa group (Fig. 5B). Upregulated proteins include Aldhla2, Apoa5 and Hspa8, etc. and downregulated proteins include Adss, Sycp3 and Gip, etc. in the 90 kPa group (Fig. 5C). Upregulated proteins include Aldoa, Nme2 and Icam1, etc. and downregulated proteins include Gip, Sycp3 and Ly6g6c, etc. in the 120 kPa group (Fig. 5D).

Coremine Medical’s ontology-based medical information retrieval platform, coupled with NCBI’s GeneBank gene database, was employed to mine protein data related to acute brain injury, explosion shock wave injury, and brain function-related phenotypes. This analysis, combined with cluster analysis, identified three co-expressed differential proteins: Aldoa and Nme2 (both upregulated) and RacGAP1 (downregulated) (Fig. 5E). To validate these findings, serum levels of Aldoa, Nme2, and RacGAP1 were measured using ELISA kits. Results revealed statistically significant differences in Aldoa and RacGAP1 levels compared to the Sham group (p < 0.05) (Fig. 5F, H). However, Nme2 levels did not show statistically significant differences between the groups (Fig. 5G). This research highlights the potential of integrating ontology-based platforms with gene databases for identifying key biomarkers related to blast-induced brain injury. Further investigation into the functional roles of Aldoa and RacGAP1 in the context of blast injury may lead to the development of novel diagnostic tools and therapeutic strategies.

Fig. 5
figure 5

Histogram of quantity distribution of differentially expressed protein (A). Volcano plot of DEPs in the test and the sham group (B, C, D). Each dot represents a protein, with red representing significantly up-regulated differentially expressed proteins, blue representing significantly down-regulated differentially expressed proteins, and gray representing non-differentially expressed proteins. Relative expression of protein in Protein-seq (E). The horizontal and vertical coordinates represent genes name and the relative expression values. Relative expression log2FC refers to the logarithmic value (base 2) for the fold change of differentially expressed genes between the test and sham group. Expression of Aldoa (F), Nme2 (G) and RacGAP1 (H) in serum was verified by ELISA. (n = 5 per group) (L means 60 kPa group, M means 90 kPa group, H means 120 kPa group, S means sham group, *p < 0.05 vs. Sham, &p < 0.05 vs. 60 kPa).

Full size image

GO and KEGG pathway analysis

Gene Ontology (GO) analysis revealed a significant enrichment of specific biological pathways in response to varying shock wave overpressure. Compared to the sham group, the 60 kPa exposure group showed enrichment in 1121 GO terms (Fig. 6A), with 121 related to cell components, 102 to molecular functions, and 898 to biological processes. The 90 kPa exposure group exhibited an even greater enrichment, with 2096 GO terms (Fig. 6B), including 248 related to cell components, 216 to molecular functions, and 1632 to biological processes. Similarly, the 120 kPa exposure group displayed significant enrichment in 1121 GO terms (Fig. 6C), with 103 related to cell components, 88 to molecular functions, and 930 to biological processes.

KEGG pathway analysis further highlighted the impact of blast exposure on cellular processes. The 60 kPa exposure group showed significant enrichment in 33 KEGG pathways (Fig. 6D), while the 90 kPa exposure group displayed enrichment in 47 pathways (Fig. 6E). The 120 kPa exposure group exhibited enrichment in 26 KEGG pathways (Fig. 6F). These findings suggest that blast exposure, at varying pressure levels, leads to widespread disruption at the cellular and molecular levels, impacting diverse biological processes.

Fig. 6
figure 6

Differential expression protein GO enrichment analysis histogram of 60 kPa (A), 90 kPa (B) and 120 kPa (C) group. The horizontal and vertical coordinate represent GO Term and the number of differentially expressed proteins. Green represents cellular component annotation information, blue represents molecular function annotation information, red represents biological process annotation information, and transparency represents p-value size. The darker the color, the smaller the p-value. Bubble diagram of KEGG metabolic pathway enrichment analysis of differentially expressed proteins in 60 kPa (D), 90 kPa (E) and 120 kPa (F) group. The horizontal and vertical coordinate represent enrichment rich factor value and KEGG pathway information. Among them, size of circle represents the number of differentially expressed proteins in the pathway, and the larger the circle, the more the number; Color of circle indicates p-value size, and the redder the color, the smaller the p-value.

Full size image

Discussion

The effects of explosive blasts on the central nervous system are an increasingly common cause of persistent and often debilitating neurological problems among both military service members and civilians. A thrust for advanced research efforts to understand the injury mechanisms and subsequent pathophysiology of blast neurotrauma are underway. The current study was conducted to determine an overpressure injury threshold for mild blast neurotrauma based on neurobehavior and biomarker changes in the rodent model. Previous studies have demonstrated that rats exposed to shock wave overpressure exhibit heightened anxiety-like behavior and cognitive function in the acute phase14,15. However, the long-term behavioral consequences of shock wave overpressure exposure in the subacute and chronic phases remain poorly understood. This research sheds light on this gap, revealing a dynamic pattern of anxiety in rats following shock wave overpressure exposure. We observed a significant increase in anxiety-like behaviors levels in the early days post-exposure, followed by a return to normal at one week. Interestingly, anxiety levels then escalated again at both one and three months. These findings suggest that overpressure of 120 kPa exposure can induce persistent anxiety-like behavior in rats, even in the subacute and chronic phases (Table 1).

The rat active avoidance experiment provides a reliable method for quantifying changes in animal behavior. A higher active avoidance sub-value indicates superior memory ability, while a shorter reaction time reflects deeper memory and a faster response16,17. In our study, we observed a unique pattern of memory impairment in rats exposed to 120 kPa blast overpressure (BOP). Specifically, we found reduced learning ability and impaired memory only in the early stages of exposure. This finding aligns with behavioral observations in rats with concussion-like brain injuries18. However, our study did not detect memory deficits at other time points, a result that contrasts with studies showing memory impairment in rats after extended intervals (> 4 months) following explosions19,20. One study reported that C57BL/6 mice require a higher blast overpressure (415 kPa) to cause impaired fear memory after a single explosion21. Collectively, these findings suggest that in both rats and mice, lower blast overpressure (60 to 120 kPa) does not significantly impair memory within the first three months following brain injury.

The neurotrophin plays an important role in the expression of anti-inflammatory factors and decreased neuronal loss22. Increased BDNF expression was observed following low level blast, therefore implying ongoing neuroprotection. However, once exposed to higher pressure level, a decrease in BDNF expression was observed, suggesting that a threshold exists. BDNF expression decreases significantly as time after injury increases, implying that brain damage may persist. 5-HT and DA are neurotransmitters that regulate anxiety, depression and other emotions and cognitive memory behaviors. They are cleared by astrocytes and released into the blood through binding with excitatory G protein coupled receptors, and play a key role in the central nervous system and mental diseases23,24. The content of 5-HT and its effect on its related receptors lead to changes in mood25,26. DA is mainly involved in emotion regulation and emotion expression through the dopamine pathways of mesbrain-cortex and mesbrain-limbic lobe27,28,29. Our results showed that 5-HT and DA were significantly reduced in the early and late stages in the medium-high level overpressure exposure group, which may be related to the occurrence of anxiety-like behaviors in rats. GABA is an important inhibitory neurotransmitter in the central and peripheral systems of the human body, mediating most of the inhibitory effects. Interestingly, no changes in serum GABA levels were observed throughout the blast exposure. The reasons for this are not clear.

Proteomic sequencing which results shed light on potential causes of neurological dysfunction following shock wave exposure offers a powerful tool for analyzing changes in protein expression levels in peripheral blood following shock wave overpressure exposure. The downregulated proteins in the 120 kPa exposure group include Gip, Sycp3, Galk1, Ddx18, Lrig3, RacGAP1 etc. The downregulated expression of Gip (glucose-dependent insulinotropic polypeptide) may lead the occurrence of neurodegenerative diseases30. Sycp3 is a potential protein involved in testicular development and spermatogenesis31,32,33. Galk1 (galactokinase 1) is the source of elevated galactose-1-phosphate and cerebrosides are modestly reduced in a mouse model of classic galactosemia34. Ddx18 was found to be a novel positive regulator that plays a role in cell proliferation and synthesis of milk protein35. Lrig3 was thought to be a gene highly implicated in the development of the nervous system36. RacGAP1 is a protein associated with cellular processes such as proliferation, growth regulation, transformation, and metastasis37,38. Studies have shown that RacGAP1 is involved in cytokinesis, induces cell proliferation, and may be linked to the proliferative marker Ki6739. Additionally, RacGAP1 has been found to play a role in cell transformation, migration, and metastasis40. The PI3K/AKT signaling pathway is a crucial cellular signaling cascade that regulates essential cellular processes, including proliferation, growth, survival, and metabolism. The current study revealed that RacGAP1 knockdown decreased the protein expression of PI3K and p-AKT, while having minimal impact on total AKT expression41. This suggests that RacGAP1 may regulate the PI3K/AKT pathway. KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies42,43,44. The results of our study show that KEGG enrichment significant pathway in the 120 kPa group are human papillomavirus infection, PI3K − Akt signaling pathway and focal adhesion. The pathogenesis of anxiety or depression is complex and involves multiple signal transduction pathways and molecular changes. The PI3K/AKT pathway is an essential signaling pathways in neurons, which is widely expressed in emotion-related regions of the brain. Therefore, the PI3K/AKT pathway may play a moderating role in mood disorders45. The PI3K/AKT pathway is involved in shaping chronic neuroinflammation in microglia and anxiety46. The study revealed that the PI3K/AKT pathway is involved in the regulation of anxiety and depression-like behaviors in mice with colitis47. Our study showed that 1d after shock wave exposure, 120 kPa rats showed anxiety-like behavior, and their serum RacGAP1 protein expression decreased, suggesting that RacGAP1 protein may be an entry point to study the mechanism of shock wave-induced anxiety-like behavior in rats.

The upregulated proteins in the 120 kPa exposure group include lcam1, Aldoa, Nme2 etc. Lcam1 is the known human gene loci associated with nonsyndromic hydrocephalus48. Glucose is the primary fuel that powers the vast synaptic activity in the human brain. Neurons require efficient metabolism of glucose through glycolysis and oxidative phosphorylation49. Alternatively, in events of brain injury, lactate can be used as an alternative source of energy50. Studies show that Aldoa is upregulated in patients with Alzheimer’s disease51,52,53. Altered glucose metabolism in the brain has long been observed as a key pathophysiological feature of AD. As our results show, the 120 kPa-exposed rats experienced learning and memory dysfunction 1 day after shock wave exposure, while their serum Aldoa levels were elevated, suggesting that Aldoa may be a potential biomarker for predicting learning and memory dysfunction caused by shock wave exposure. In addition, aldoa, acting as a glycolysis monitor, plays a critical role in regulating NLRP3 inflammasome activation by sensing changes in glycolysis flux54. This suggests that Aldoa may be involved in the inflammatory response following brain injury. Inhibiting Aldoa activity could potentially provide a therapeutic strategy for suppressing inflammation after brain injury. These findings could contribute to the development of novel diagnostic and therapeutic strategies for shock wave overpressure related brain injuries. Further research is needed to fully elucidate the specific roles of these proteins and their potential therapeutic targets. The identification of these differentially expressed proteins, Aldoa and RacGAP1, provides valuable insights into the mechanisms underlying brain injury induced by acute shock wave overpressure exposure.

A finding with our dataset was that the high-level pressure exposure demonstrated to be the most injurious. The data indicated that a pressure intensity of 120 kPa produced the overall greatest neurological changes as compared to sham animals. This finding aligns with the research results of Pamela J11. In this study, we evaluated the neurobehavior at acute (1 d, 7 d) and more chronic time points (28 d and 90 d) after shock wave exposure, with the aim of gaining a systematic understanding of the changes in mood and cognitive function at different times after shock wave exposure. At the same time, through the analysis of the serum proteome in the acute phase, biomarkers that can quickly characterize and predict brain injury in the early stage after shock wave exposure were screened, in order to make judgments and interventions in the shortest time after shock wave exposure. Neurological complications following a blast injury, including depression, anxiety, and memory problems, often persist even when brain damage is undetectable. Research shows that highly controlled detonations of small, 1.7-gram 1,3,5-trinitro-1,3,5-triazinane (RDX) spherical charges reduced synaptic markers known to be downregulated in cognitive disorders, but without causing overt neuronal loss or astroglial responses55. Further alarming are the numbers of blast-exposed individuals returning from war zones with no detectable physical injury or neuropathology, but who still suffer from persistent neurological symptoms including depression, anxiety, sleep disturbances, and memory problems. Delayed cellular responses occur due to blast exposure56, suggesting that a subset of exposed individuals can have no apparent injury or symptoms, only to experience delayed effects to brain mechanisms and behavioral functions. Timely identification and proper managing of blast‐induced brain trauma are critical since traumatic brain injuries (TBIs) often worsen in clinical outcome within 48 h if not treated appropriately4. Those with blast‐induced neurotrauma and associated neurological intrusion, but without the typical neuropathology of a TBI, face a major challenge for diagnosis. Neuroimaging techniques are improving but have yet to achieve the level of sensitivity needed to detect subtle blast‐induced alterations that underlie lasting neurological impediments. Peripheral blood biomarkers could be used as signals to evaluate brain injury and the efficacy of specific pharmacological treatments that could delay, stop, or reverse possible pathological proteins in the brain related to exposure to blast57.

Limitations and strengths

This study offers valuable insights into neurobehavioral and serum protein changes following varied blast magnitudes across acute and chronic phases. However, a notable limitation is the lack of focus on protein changes within brain tissue, specifically the hippocampus. Future research should include an examination of these changes in the hippocampus, as this region is crucial for learning, memory, and spatial navigation, and is often affected by blast injuries. Additionally, increasing sample sizes would further bolster statistical significance and deepen our understanding of post-injury phenomena. Despite these limitations, the current study provides a solid foundation for future investigations into the mechanisms underlying shock wave overpressure exposure.

Methods

Animals and ethics approval

Male Sprague–Dawley rats, aged 6 weeks and weighing 180–220 g, were procured from SPF (Beijing) Biotechnology Co., Ltd. The feeding of rats and all experimental procedures were carried out in accordance with the protocols of the Animal Ethics Committee of the Technical Research Center for Toxicology and Biological Effects of the Ordnance Industry Health Institute and approved by it. Prior to the experiments, all the rats were housed in specific pathogen-free facilities. Five rats were kept in cages, given paper towel and sawdust, and given free access to food and water.

Shock wave exposure

A total of 192 rats were divided into 60 kPa, 90 kPa, 120 kPa group and sham group, with 48 rats in each group. Blast exposures were performed at the laboratory with a biological shock tube (BST). Figure 1A is the schematic of a biological shock tube. The biological shock tube is composed of high-pressure driver section, diaphragm and low-pressure driver section. The high-pressure section is about 0.4 m long, with a cylindrical section and an inner diameter of 100 mm. The aluminum diaphragm is located between the high-pressure tube and the low-pressure tube. The total length of the low-pressure tube is about 2.5 m, with a square section. Side length is 88.6 mm. There is an observation light window in the low-pressure section for observing the effector. The driver was sealed by the membrane material and pressurized to 60–120 kPa, before bursting the membrane. For shock-wave exposure (1 Blast, *60–120 kPa peak pressure, recorded from a pressure sensor positioned adjacent to the animal), rats were anesthetized with 3% isoflurane in 100% oxygen for 4–6 min and placed in a thoracic and abdominal protective device, with their ventral side facing the blast source. The rats in the sham group received only anesthesia and noise exposure, but not shock wave overpressure exposure. To ensure quality, the pressure sensor used in this study was PCB piezoelectric type (Depew, NY, 113B28). All data were sampled and recorded at a frequency of 2.0 MHz, with a single acquisition time of 200 ms. The pressure we measure is the passing pressure, and the sensor is perpendicular to the direction of shock wave propagation.

Table 1 Test matrix indicating exposure pressures, time and the number of animals in which the rats were tested following exposure.
Full size table

Behavioral evaluation

A series of behavioral experiments were performed on 1 d, 7 d, 28 d and 90 d after explosion (n = 12). During the week before the test, all the rats were kept in specific rooms to adapt to the experimenter and operating environment. All tests were conducted between 9 a.m. and 6 p.m. All data were detected by an experimenter unaware of the grouping using automatic video tracking software (Serirs30000 Conditional Behavioral Analysis System, German TSE).

Anxiety-like behaviors were evaluated by light–dark box test and open field test. According to standard procedure, the rats were placed in light–dark box and open field for five minutes respectively. In the light–dark box test, number of visits and time spent in light were recorded. Distance traveled and number of visits in center were recorded in the open field experiment.

The function of learning and memory was evaluated by two-way active avoidance experiment. The experiment consists of two parts: training process (learning) and testing process (memory). Training process: the rats were placed in one compartment and subjected to 40 active avoidance experiments for 4 consecutive days. At the beginning of the experiment, the rats have a 30 s adaptation period. After the software automatically detected the rat’s position, the rats were given acoustic stimulus for 10 s (conditioned reflex). If the rats did not shuttle to the opposite compartment within 10 s (active avoidance period), the corresponding compartment was subjected to 1.0 mA of sole electric stimulus (unconditioned reflex), and the sole electric stimulus duration was 3 s. After 3 s of sole electric stimulus, the sound stimulus still exists, the escape shuttle response will terminate the electric and sound stimulus during this time. If the shuttle response does not occur within 13 s, the escape fails. The fifth day is test period: the other steps are same as the acquisition period, except that plantar electric shock is not given. Active avoidance times and response time were recorded.

After the end of the experiment, researchers need to clean up the feces of each rat and use 75% ethanol solution to eliminate the odor left by the rat in the box to avoid interference with the next rat.

Serological examination

At 1 d, 7 d, 28 d and 90 d post injury (n = 5), rats were deeply anesthetized with 2% pentobarbital sodium (50 mg/kg, intraperitoneal injection). Peripheral blood of the rats in the four groups was taken and serum was isolated from the blood. Serum samples were stored at − 80℃ for future analysis. Serum levels of BDNF, 5-HT, DA and GABA were detected by ELISA kit in rats (Enzyme-linked Biotechnology, Shanghai, China).

Proteomic analysis

At 24 h post injury, peripheral blood serum (n = 5) of rats was collected and protein was extracted. The protein concentration of the corresponding sample was calculated by BCA quantitative method. Samples were prepared by acetone precipitation, protein proteolysis, reduction, alkylation modification and proteolytic hydrolysis. The total peptide of each sample was separated by the nano-UPLC liquid phase system EASY-nLC1200, and the sequencing data was collected by Q-Exactive HFX equipped with nanoliter ion source. p-value or p-value-chitest < 0.05 was used as the screening condition, and the foldchange ≥ 1.5 or ≤ 0.67 was used as the significantly up-regulated or down-regulated differentially expressed proteins. Protein FDR was set to 1% FDR. The screened differentially expressed proteins were confirmed by enzyme-linked immunosorbent assay. The main enrichment biological functions of differential proteins were determined by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.

Statistical analysis

Results were presented as the mean ± standard deviation. Statistical analysis and mapping were performed according to GraphPad Prism 8.0 software. Differences between multiple groups were tested by one-way ANOVA. Tukey’s multiple comparisons test was used for multiple comparisons. Data in the training process of the two-way active avoidance experiment were compared using repeated measures analysis of variance. Statistical significance was established as p < 0.05.