Impact of blast exposure on visual pathway: Mechanism exploration and novel diagnostic perspectives
Yue Wang, Nan Yang, Xiaofan Chen, Xing Chen, Yalei Ning, Rongdi Yuan
TL;DR
This study explores how blast exposure causes visual problems in mice, revealing two phases of damage and a potential treatment to prevent long-term vision loss.
Contribution
The study identifies biphasic visual dysfunction after blast exposure and proposes NLRP3 inflammasome targeting as a novel therapeutic strategy.
Findings
Visual dysfunction emerged at 24 hours post-blast and reappeared at 28 days, indicating a biphasic pattern.
Early optic nerve demyelination and later visual cortex neuronal pyroptosis were key pathological features.
MCC950 treatment reduced neuroinflammation and prevented late-phase visual impairment.
Abstract
Blast-induced traumatic brain injury (bTBI), frequently observed in modern warfare, often presents without overt clinical symptoms initially, yet can involve visual impairment. However, the underlying mechanisms and long-term outcomes of visual dysfunction following blast exposure (BE) remain poorly understood. This study aimed to investigate the potential delayed effects of BE on visual function. A bTBI mouse model was established using a biological shock tube. Neurological deficits were assessed via the modified neurological severity score, while visual function was evaluated at multiple time points using flash visual evoked potentials (F-VEP) and a light-dark shuttle box. Ultrastructural evidence of damage was obtained through transmission electron microscopy (TEM). Inflammatory and pyroptosis markers were localized and quantified via immunofluorescence staining and Western blotting.…
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Taxonomy
TopicsTraumatic Brain Injury Research · Traumatic Ocular and Foreign Body Injuries · Traumatic Brain Injury and Neurovascular Disturbances
Introduction
Blast-induced traumatic brain injury (bTBI) is a specific type of neurotrauma resulting from exposure to high-speed and high-pressure shock waves that propagate radially from the epicenter of an explosion [1,2]. While this pathological condition can occasionally be observed in civilian settings following chemical or industrial accidents [3], it is predominantly prevalent in military contexts, where it has emerged as a signature injury of modern warfare [4,5]. According to reports, the estimated number of bTBI cases sustained during the conflicts in Iraq and Afghanistan was as high as 320,000 cases [6]. Although it has been demonstrated that bTBI results in systemic damage via multiple distinct pathophysiological mechanisms. Mild traumatic brain injury (mTBI) accounts for a relatively high proportion of bTBI cases [1]. However, mTBI may not exhibit obvious clinical manifestations during the initial phase and remains particularly challenging in clinical diagnosis [7].
The eyes, often referred to as the “window to the brain,” serve as a potential entry point for craniocerebral blast injury due to their direct exposure to shock waves during explosive events [8]. The visual system, as a critical component of the central nervous system (CNS), depends on intricate and precisely organized neural circuits to maintain the integrity of its functions [9]. Blast overpressure is usually complicated by visual dysfunction through injury to several neuroanatomical pathways, including the visual cortex, brainstem structures, optic nerves, or direct eyeball injuries [8]. Longitudinal clinical studies have consistently demonstrated that survivors of bTBI usually suffer from poor visual quality, characterized by visual field defects and diminished contrast sensitivity [10]. It indicates that there may be delayed visual dysfunction after blast exposure (BE). Due to the close anatomical and developmental relationships between the visual system, including the retina and its projection system, and the CNS, exploring visual dysfunction after BE is of great significance.
So far, research on bTBI-related visual dysfunction has predominantly focused on retinal ganglion cell (RGC) damage within the extracranial segment of the visual pathway [11–15]. However, the injury mechanisms and functional consequences of the intracranial segment of the visual pathway, as a crucial route for visual information processing, remain unclear after BE. This gap substantially limits the comprehensive understanding of the pathological mechanisms underlying bTBI-associated visual dysfunction. Notably, neuropathological processes, including microvascular damage, axonal injury, and neuroinflammation, usually emerge within a timeframe extending from several weeks to years post exposure, potentially leading to long-term effects on visual function, cognitive performance, and emotional well-being [10,16].
Neuroinflammation has been identified as a critical pathological response and a key mechanism underlying these adverse effects [17]. The nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is a key sensor within the innate immune system. Upon detection of exogenous pathogenic invasions or endogenous cellular damage is recognized, an inflammatory response is initiated through the assembly of the NLRP3 inflammasome [18], which consists of three core components: NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1 [19]. These components act synergistically to regulate the specificity and activity of inflammasomes, the maturation of cytokines IL-1β and IL-18, and the processing of Gasdermin D, thereby promoting cytokine release and pyroptosis—a distinct form of lytic, inflammatory programmed cell death [20,21]. Previous studies have demonstrated that the p38/MAPK and AKT/NFκB signaling pathways are key mediators in neuroinflammatory processes and essential upstream regulators of NLRP3 inflammasome activation [22,23]. Pharmacological modulation of NLRP3 inflammasome activity has become a promising therapeutic strategy, showing remarkable efficacy in preventing a fundamental molecular mechanism for preventing the progression of neuroinflammatory diseases [24,25].
This study aims to investigate the dynamic changes of visual dysfunction in a bTBI mouse model and the potential impact of optic nerve demyelination and NLRP3 mediated neuronal pyroptosis in the visual cortex on visual dysfunction after shock wave exposure. Furthermore, we explore the therapeutic potential of regulating the NLRP3 inflammasome for visual dysfunction in the bTBI mouse model. The findings of this work provide new evidence for understanding the delayed visual function impairment associated with bTBI, and a new perspective for the auxiliary assessment and monitoring of bTBI through non-invasive visual electrophysiological examination.
Methods
Animal
Adult male C57BL/6J mice (6–8 weeks old) were obtained from the Experimental Animal Center of Daping Hospital, Army Medical University (Certificate No. SCXK [Yu] 2002–0002, Chongqing, China). Following a 7-day acclimatization period in the isolation room, the animals were housed under standardized conditions with controlled temperature (22 ± 1 ℃) and humidity (55 ± 5%), maintained on a 12-hour light/dark cycle. The mice were provided with ad libitum access to autoclaved water and standard laboratory chow throughout the experimental period. Animal research in this study was conducted in accordance with the ARRIVE guidelines (https://arriveguidelines.org). The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Army Medical University (Approval No. AMUWEC-20199022). All surgical procedures were carried out under tribromoethanol anesthesia (250 mg/kg, intraperitoneal injection, i.p.), with strict adherence to humane principles to ensure the highest standard of animal welfare.
Establishment of bTBI mouse model
The bTBI model was established using a biological shock tube-1 (BST-1) apparatus [26], as illustrated in S1A in S1 Fig. The experimental protocol was modified from previously described methods [27]. Male C57BL/6J mice were randomly assigned to the bTBI group or the sham group. Prior to BE, mice were anesthetized with tribromoethanol (250 mg/kg, i.p.) and securely positioned in a custom-designed metal restraint frame to minimize movement during BE and prevent secondary displacement injuries. All restraint frames were aligned in the same vertical plane relative to the BST-1 biological shock tube to ensure consistent pressure exposure. The blast wave was generated by rapid rupture of an aluminum diaphragm using high-pressure compressed gas, producing a peak overpressure of 354 ± 3.47 kPa with a duration of 0.05 ± 0.01 s (S1B in S1 Fig), simulating open-field conditions for bTBI induction. Sham-operated animals underwent identical anesthesia and positioning procedures but were not exposed to the blast wave. Following BE, all the animals were monitored until they fully recovered consciousness before being returned at the housing facility. Electrophysiological and behavioral tests were conducted at predetermined time points (24 hours, 3, 7, and 28 days after exposure), after which the mice were euthanized by cervical dislocation under anesthesia induced by tribromoethanol (250 mg/kg, i.p.). Subsequently, samples were collected for subsequent experimental analysis. Furthermore, animal health and behavior were monitored at least twice daily. Any mouse demonstrating an inability to eat or drink, a loss of > 20% of baseline body weight, or signs of severe distress (e.g., persistent convulsions, severe tremors, or paralysis) were humanely euthanized prior to the scheduled endpoint via cervical dislocation following deep anesthesia with tribromoethanol (250 mg/kg, i.p.). In the present experiment, all animals remained healthy and survived to the planned experimental endpoint. No animals met the predefined humane endpoint criteria, and no unscheduled deaths occurred.
Modified neurological severity score (mNSS)
The modified neurological severity score was conducted 24 hours post BE (n = 30). The specific operational steps were based on previous studies [28]. In brief, the mNSS includes 10 tasks, which can assess the motor function (muscle status and abnormal movement), sensory (vision, touch and proprioception), balance and dull/sharp stimulus reflex functions of mice. The score was graded from 0 (normal) to 18 (maximal deficit score). One point is given for each abnormal behavior or the absence of a test reflex. Therefore, the higher the score, the more severe the neurological injury. The neurological score assessment was conducted by two experienced researchers in a quiet environment in a well-lit room, without knowing the experimental treatment. Similarly, all the following analyses were conducted by researchers who were unaware of the injury group.
Flash Visual Evoked Potential (F-VEP)
The F-VEP recording protocol was adapted from previously established methods with modifications [29]. Following an 8-hour dark adaptation period, mice were anesthetized via intraperitoneal administration of a sodium pentobarbital (50 mg/kg). Animals were then secured in a stereotaxic frame, and F-VEP recordings were initiated 15 minutes post-anesthesia induction to ensure stable anesthetic depth. The three-electrode system was implemented for optimal signal acquisition. A platinum needle electrode was inserted subcutaneously over the visual cortex served as recording electrode (1.5 mm laterally to the midline, 1.5 mm anterior to the lambda), reference electrode (behind the ear on the same side), and ground electrode (tail). During monocular testing, the contralateral eye was occluded using a light-proof patch to prevent cross-stimulation. Each recording session consisted of 54 consecutive flash stimuli (10 ms duration, 1 Hz frequency) with an intensity range of 120–200 mJ [30]. The sample sizes were n = 30 for the BE 24h, 3d, 7d, and 28d groups, and n = 12 for the continuous F-VEP recording group. For the drug treatment experiments, the sample size was n = 20 per group at each time point. Based on previous studies demonstrating the superior stability of P2 wave parameters [31], we focused our quantitative analysis on two key metrics: (1) P2 wave latency and (2) N2-P2 amplitude. All measurements were normalized to baseline F-VEP recordings obtained prior to model establishment. Data acquisition and analysis were performed using specialized software (RetiMINERTM, China) by investigators blinded to experimental conditions.
Light-dark shuttle box
Based on the inherent preference of mice for dark environments, visual contrast sensitivity was evaluated at 24 hours and 28 days post BE using a modified light-dark box paradigm, adapted from previously established protocols [32]. The sample size was n = 14 per group at each time point. The behavioral apparatus consisted of two equally sized chambers (20 × 20 × 20 cm each) connected by a central doorway (5 × 5 cm) that permitted free movement between compartments. The testing apparatus was equipped with a high-resolution CCD camera mounted 45 cm above the chambers for comprehensive behavioral monitoring. The boundaries of the two chambers were defined using the animal movement trajectory tracking system EthoVision® XT (Noldus Information Technology, Wageningen, Netherlands), and animal movement was tracked and analyzed also using it. The system’s infrared sensors and advanced image processing algorithms enabled precise determination of animal position and movement patterns. Each mouse underwent a 5-minute acclimatization period followed by a 5-minute testing session. The tracking system recorded several parameters: (1) total time spent in each chamber, (2) the movement trajectories of experimental animals, and (3) number of transitions between chambers. The movement trajectory heat map was generated using EthoVision® XT. All behavioral tests were conducted during the light phase of the circadian cycle (09:00–17:00).
Immunofluorescence Assay
Under deep anesthesia, mice were perfused with ice-cold phosphate-buffered saline (PBS) through the heart, followed by 4% paraformaldehyde (PFA) in PBS (pH 7.4) for fixation. The sample size was n = 4 per group at each time point. Harvested brains were post-fixed in 4% PFA at 4 ℃ overnight, then dehydrated successively in 20% and 30% sucrose solutions at 4 ℃. Tissues were embedded in optimal cutting temperature (OCT) compound and sectioned coronally at 12 μm thickness using a cryostat (Leica CM1950). Tissue sections were washed with PBS and permeabilized with 0.4% Triton X-100 in PBS. After blocking with 10% normal goat serum at 37 ℃ for 1 hour, sections were incubated with primary antibodies at 4 ℃ overnight. The following primary antibodies were used: Myelin Basic Protein (MBP) (D8X4Q) XP® Rabbit mAb (1:250, CST, # 78896); βIII Tubulin Mouse monoclonal antibody (1: 300, Abcam, #ab78078), NeuN (E4M5P) Mouse mAb (1:250, CST, # 94403), Iba-1 (E4O4W) XP® Rabbit mAb (1:250, CST, # 17198), and NLRP3 Monoclonal Antibody (1:100, Invitrogen, # MA5−23919). After three PBS washes, sections were incubated with species-appropriate Alexa Fluor®-conjugated secondary antibodies (488/594/647, 1:450, CST) at 37 ℃ for 1 hour. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Fluorescence images were captured using a confocal microscope (Leica TCS SP8) with consistent acquisition parameters across samples. Sections were mounted with anti-fade mounting medium and stored at 4 ℃. Quantitative analysis of immunofluorescence signals was performed using ImageJ software.
TUNEL assay
The sample preparation and tissue sectioning procedures were conducted as previously described. The sample size was n = 4 per group at each time point. Brain sections were subjected to double staining using NeuN antibody and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay kit (Beyotime, #C1090) to identify DNA damage, following the manufacturer’s protocol. Specifically, after incubation with the NeuN primary antibody and fluorescent secondary antibody, the sections were reacted with the TUNEL reaction mixture at 37 ℃ for 1 hour. Subsequently, nuclear staining was performed using 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Fluorescence images were captured using a confocal microscope (Leica TCS SP8) with consistent acquisition parameters across samples.
Western blotting
Protein expression was analyzed via Western blotting as previously described [33]. The sample size was n = 3 per group at each time point. Specifically, brain tissues were homogenized in RIPA lysis buffer (Beyotime) supplemented with protease and phosphatase inhibitors. Protein concentrations were quantified using a BCA protein assay kit (Solarbio) according to the manufacturer’s protocol. Equivalent amounts of protein samples were resolved by SDS-PAGE on gels of appropriate concentration (Bio-Rad), selected based on the molecular weights of the target proteins, followed by electrophoretic transfer onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with 5% non-fat milk in TBST for 1 hour at room temperature to block non-specific binding sites. Subsequently, the membranes were incubated overnight at 4℃ with gentle agitation using the following primary antibodies: anti-MBP (1:10,000; CST) anti-Iba-1 (1:10,000; CST), anti-NLRP3 (1:10,000; Invitrogen), anti-IL-1β (1:8,000; CST, # 12242) anti-cleaved Gasdermin D (1:10,000, CST, # 10173), and anti-GAPDH (1:10,000, Abcam, # ab9485). Total protein levels were quantified using stain-free technology on the ChemiDoc imaging system (Bio-Rad) for normalization. Following five washes with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were detected using enhanced chemiluminescence and visualized with the ChemiDoc imaging system (Bio-Rad). Quantitative analysis of band intensity was conducted using Image Lab software (Bio-Rad), with GAPDH serving as the loading control. All experiments were performed in triplicate to ensure reproducibility and reliability.
Transmission electron microscopy (TEM)
Optic nerve tissues were fixed overnight in 2.5% glutaraldehyde prepared in phosphate buffered saline (PBS, pH 7.4), followed by post fixation with 1% osmium tetroxide for 2 hours at room temperature. They were dehydrated in a graded series of ethanol solutions and propylene oxide, impregnated with a mixture of (1:1) propylene oxide/Epon resin (Sigma), and then embedded in pure epoxy resin, which was allowed to polymerise for 48 h at 60 ℃. Slicing with LKB-I ultramicrotome (LKB, Sweden) and mounted on 200 mesh grids. The sections were then double stained with uranyl acetate and lead citrate before being photographed using a Hitachi TEM system (zoom-1, HC-1) operated at an accelerating voltage of 80.0 kV.
Drug Treatment
MCC950 (HY-12815A, MCE, USA) was administered as previously described [20,34]. The drug solution was prepared according to the manufacturer’s protocol by dissolving MCC950 in a vehicle consisting of 5% dimethyl sulfoxide (DMSO) and 95% PBS. This pharmacological intervention aims to investigate the effects of MCC950 on the visual function in mice following BE. bTBI mouse models randomly assigned to two experimental groups: the bTBI + DMSO group (solvent control group) and the bTBI + MCC950 group (treatment group) (S2 Fig). For the BE24 h group, MCC950 (*i.p.*10 mg/kg) was injected on days 0 and 1 post exposure. While for the BE28 d group, MCC950 (*i.p.*10 mg/kg) was injected on days 0, 1, and 2 post exposure, followed by injections every 2 days for a total of six administrations. The mice in the solvent control groups received equivalent volumes of solvent (5% DMSO in PBS) at the corresponding time points. After electrophysiological and behavioral assessments were completed, the animals were euthanized under anesthesia at the corresponding time points. The samples were collected for subsequent experimental analysis.
Statistical analysis
All data in the charts are presented as the mean ± standard deviation (SD), with each point representing the data of an individual experimental animal. Repeated measures ANOVA was used to analyze F-VEP values measured at multiple time points, while one-way ANOVA followed by Duncan’s multiple range test was employed for other between-group comparisons. p value < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 23 software (SPSS, Chicago, IL, USA).
Results
Neurobehavioral evaluation of the bTBI mouse model
To evaluate the neurological deficits in the bTBI mouse models, at BE 24h, mNSS assessments were conducted. The results showed minimum score of 0, a median score of 3, and maximum score of 6 are summarized in Table 1. This result indicates that blast exposure successfully induced neurological dysfunction in mice consistent with mTBI degree. Subsequently, mice were randomly assigned to each experimental group.
Table 1: Distribution and proportion of mNSS scores of mice post BE.
Temporal progression of blast-induced visual function disorder: acute dysfunction at 24 hours, transient recovery, and secondary decline at 28 days
Following establishing a bTBI mouse model, F-VEP were recorded at multiple time points post BE, including 24 hours, 3 days, 7 days, and 28 days, to evaluate the temporal alterations in visual pathway function (Fig 1A-C).These parameters were highly reproducible in F-VEP recordings from 6-8-week-old healthy C57BL/6J mice (sham group), with a P2 latency of 69.62 ± 6.13 ms (Fig 1D) and a P2 amplitude of 6.62 ± 1.25 μV (Fig 1E). Compared to the sham group, the BE 24 h group exhibited a significantly prolonged P2 latency (p < 0.001) and reduced N2-P2 amplitude (p < 0.001) at BE 24 h group (Fig 1D-1E). At 3 days post exposure, F-VEP showed that compared to the BE 24 h group, a reduction in P2 latency and an increase in N2-P2 amplitude were observed; however, these changes did not reach statistical significance (Fig 1D-1E). At 7 days post BE, F-VEP results showed no significant differences compared to the sham group (Fig 1D-1E), indicating a gradual recovery of reaction speed and conduction ability in the visual pathway; visual function had essentially returned to pre-injury levels. At 14 days post exposure, re-examination revealed a decline in visual function in some mice. However, this decline was not statistically significant. By 28 days post BE, the bTBI model mice exhibited a consistent and significant prolongation of the P2 latency (p < 0.01) as well as a significant reduction in the N2-P2 amplitude (p < 0.001) (Fig 1D-1E). Further analysis at 42 days and 56 days post BE revealed persistent visual dysfunction, with no significant changes compared to the 28-day time point (S3 Fig). In addition, we conducted longitudinal F-VEP recordings on bTBI mice to observe the pre- and post-changes in individual visual function. The results were consistent with previous observations. These mice experienced a decline in visual function 24 hours post BE, followed by a transient recovery period, and then visual dysfunction again 28 days post BE (Fig 1F). These electrophysiological alterations suggest that the biphasic decline in visual function observed following blast exposure might indicate damage to the functionality of the visual pathway. Therefore, we selected 24 hours and 28 days post exposure as the critical time points for assessing visual function disorders and subsequent experiments.
Electrophysiological and behavioral assessments of visual dysfunction following blast wave exposure.(A) Schematic illustration of the preparation procedure and observation time points for the bTBI animal model. (B) Schematic diagram of F-VEP recording device (C) Representative F-VEP waveforms at BE 24h, BE 3d, BE 7d, and BE 28d, the horizontal grid is 50 ms and the vertical grid is 10 μV. Quantitative analysis of P2 latency (D) and N2-P2 amplitude (E) (n = 30). (F) Continuous F-VEP recording (n = 12), the shaded area indicates mean ± SD. (G) Schematic of the bright chamber and dark chamber in the Light-dark box test. (H) Heatmap (left figure) and trajectory (right figure) visualization of mouse positions in different groups. (I) Quantitative analysis of stay time in dark areas in different groups (n = 14). Error bars indicate mean ± SD. ** p < 0.01, *** p < 0.001, ns p > 0.05.
We conducted the light-dark shuttle box at two key time points identified with visual dysfunction to further evaluate light-dark discrimination ability (Fig 1G). We observed a distinct spatial distribution pattern. Mice in the sham group showed a strong preference for the dark compartment, as evidenced by their frequent presence in this area on the heat map. In contrast, the BE 24 h group and the BE 28 d group exhibited a reduced preference for the dark compartment, with nearly equal distribution between the light and dark chambers (Fig 1H). Quantitative analysis revealed that the time spent in the dark compartment by the BE 24 h group and the BE 28 d group was significantly less than that of the sham group (p < 0.01) (Fig 1I). These behavioral findings are consistent with our electrophysiological data, providing strong evidence for impaired light-dark discrimination ability at 24 hours and 28 days post BE, indicating visual function deficits.
Temporal progression of blast-induced optic nerve demyelination: early onset at 24 hours and sustained progression through 28 days
To investigate whether the delayed P2 latency observed in the bTBI mouse model is associated with extracranial visual pathway injury, we selected the midsection of the optic nerve as the primary region of interest (Fig 2A). Quantitative analysis using multiplex immunofluorescence assays revealed a significant reduction in MBP fluorescence intensity within the optic nerve, accompanied by a marked decrease in the density of myelinated axons in the BE 24 h, BE 3 d, BE 7 d, and BE 28 d groups compared to the Sham group (p < 0.001) (Fig 2B-2C). Similarly, Western blot analysis confirmed a significant decrease in the MBP expression levels in all experimental groups compared with that in the sham group (p < 0.001) (Fig 2D-2E). To further investigate the ultrastructural effects of blast-induced visual dysfunction, we conducted TEM analysis of the optic nerve at two critical time points. At 24 hours post exposure, TEM showed destruction of myelin structure: significant axonal demyelination was observed, though axonal density remained relatively unchanged. However, at 28 days post exposure, the demyelination process had progressed markedly, and was characterized by widened axonal spaces and reduced axonal density (Fig 2F). These findings collectively demonstrate that demyelination of the optic nerve was evident as early as 24 h and significantly worsened 28 days post BE.
*Temporal Progression of Blast-Induced Optic Nerve Demyelination.(A) Schematic diagram of the visual pathway and key observation areas in its extracranial segment. (B) Representative images of immunofluorescence staining for MBP (green) and βIII-Tubulin (red) in mouse optic nerve slices. Group information is shown in images, scale bar = 20 μm. (C) Optic nerve relative fluorescence intensity of MBP +/β III Tubulin + between the sham group and BE 24h, BE 3d, BE 7d, BE 28d groups, n = 4. (D-E) Western blot analysis of MBP in optic nerve lysates. The molecular mass is indicated in kilodaltons, n = 3. (F) TEM analysis of the optic nerve between the sham group and BE 24h and BE 28d. Red stars indicate axons with obvious demyelination. Error bars indicate mean ± SD. **p < 0.001.
Temporal progression of blast-induced pyroptosis in visual cortex neurons: inflammatory activation emerges at 24 hours and progressively leads to neuronal pyroptosis by 28 days
We examined post BE alterations in the primary visual cortex (V1) using immunofluorescence staining and Western blot analysis. (Fig 3A). Following BE, we observed a transformation in microglial phenotype, characterized by shortened processes and a shift from ramified to amoeboid morphology (Fig 3B). Immunofluorescence analysis revealed a significant increase in Iba-1 + microglia numbers in the BE 24 h group, BE 3 d group, BE 7 d group, and BE 28 d group compared to the Sham group (p < 0.001) (Fig 3C), which was consistent with the quantitative results of Iba-1 protein levels obtained through Western blot analysis (Fig 3D, 3F). Morphological examination revealed a change in the microglial phenotype, indicating the transition of microglia from a resting state of immune surveillance to an activated state with phagocytic capabilities. These findings suggest that microglia are actively responding to pathological stimuli in the brain and executing their immunological functions [35,36]. Simultaneously, quantitative protein analysis revealed a significant upregulation of NLRP3 expression in the visual cortex (p < 0.01) (Fig 3E-3G). Immunofluorescence revealed that NLRP3 expression was predominantly localized in Iba-1 + microglia and NeuN+ neurons (Fig 3B). Additionally, statistical analysis indicated that the co-expression level of NLRP3 with NeuN was significantly elevated (Fig 3H). NLRP3 inflammasome activation triggers the release of elevated levels of IL-1β and IL-18, exacerbating neuroinflammation [37]. We subsequently evaluated the expression of IL-1β in the visual cortex by Western blotting, which indicated a significant increase post BE (Fig 3F, 3I). Moreover, we observed that expression of the pore-forming protein cleaved Gasdermin D progressively increased (p < 0.05) (Fig 3F, 3J). In short, the expression of the pyroptosis markers NLRP3, IL-1β, and cleaved Gasdermin D significantly increased, and TUNEL and NeuN colocalization markedly increased (p < 0.001) (Fig 3K, 3L). In addition, the population of NeuN+ neurons significantly decreased (p < 0.001) (Fig 3M). Collectively, these results suggest that progressive neuroinflammation in the visual cortex contributes to neuronal pyroptosis.
Temporal Progression of Blast-Induced Pyroptosis in Visual Cortex Neurons.(A) Schematic diagram of the visual pathway and key observation areas in its intracranial segment. (B) Representative images of immunofluorescence staining for NeuN (green) and NLRP3 (red), Iba-1 (grey) in mouse visual cortex slices, Group information is shown in images. The red triangle marks indicate the co-localization of NLRP3 and NeuN. The scale bars for all fluorescence intensity channels and the merge are set at 50 μm, the magnified inset is 20μm. Quantitative analysis of visual cortex immunofluorescence density in Iba-1 (C) and NLRP3 (G). (H) Fluorescence intensity of NeuN+NLRP3+/ NeuN+, n = 4. (D, E, F, I, J) Western blot analysis of Iba-1, IL-1β, cleaved Gasdermin D and NLRP3 in visual cortex lysates, the molecular mass is indicated in kilodaltons, n = 3. (K-L) Quantitative analysis of representative fluorescence images of NeuN staining with TUNEL labeling in the peri-contusion region from different groups that information is shown in images, Scale bar = 100 μm, n = 4. (M) Quantitative analysis of visual cortex NeuN+ cells, n = 4. Error bars indicate mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, ns p > 0.05.
MCC950 attenuates blast-induced visual dysfunction at 28 days post exposure
Visual function was assessed at 24 hours and 28 days post BE following administration of MCC950.The F-VEP results are presented in Fig 4A-4C. The F-VEP results revealed no significant differences in P2 latency and N2-P2 amplitude among the BE 24 h, BE 24 h + DMSO, and BE 24 h + MCC950 groups (p > 0.05), suggesting that MCC950 administration failed to attenuate visual impairment at the BE 24 h. Compared with the BE 28 d and BE 28 d + DMSO groups, the BE 28 d + MCC950 group demonstrated significantly preserved P2 latency and maintained N2-P2 amplitude (Fig 4D-4F, p < 0.001). The light-dark box test revealed that the distribution in the light and dark chambers among the BE 24 h, BE 24 h + DMSO, and BE 24 h + MCC950 groups was nearly equal. No apparent dark/light preference was observed in any treatment group (Fig 4G, 4H). However, at 28 days post BE, the MCC950-treated group exhibited significant behavioral changes, with a significantly increased time spent in the dark chamber compared to the BE 28 d group and BE 28 d + DMSO groups (p < 0.001) (Fig 4G, 4I). These findings indicate that MCC950 treatment effectively preserved visual function and light-dark discrimination ability in bTBI mice model at the 28 days post BE. In our systematic investigation of visual function, we conducted rigorous validation experiments to assess potential confounding factors. Comparative analysis revealed that DMSO vehicle treatment exerted no significant impact on either electrophysiological parameters or behavioral outcomes (p > 0.05, Fig 4A-4I). This critical validation enabled us to streamline our experimental design for subsequent investigations, focusing specifically on comparative analyses between the BE group and BE + MCC950 group.
*MCC950 Attenuates Blast-Induced Visual Dysfunction.(A) Representative F-VEP waveforms between the BE 24h, BE 24h + DMSO, and BE 24h + MCC950 groups, the horizontal grid is 50 ms and the vertical grid is 10 μV. (B-C) Quantitative analysis of P2 latency and N2-P2 amplitude (n = 20). (D) Representative F-VEP waveforms between the BE 28d, BE 28d + DMSO, and BE 28d + MCC950 groups. (E-F) Quantitative analysis of P2 latency and N2-P2 amplitude (n = 20). (G) Representative heatmap (left figure) and trajectory (right figure) visualization of mouse positions in different groups. (H-I) Quantitative analysis of stay time in dark areas in different groups (n = 12). All group information is shown in images. Error bars indicate mean ± SD. **p < 0.001, ns p > 0.05.
MCC950 attenuates neuroinflammation and neuronal pyroptosis in the visual cortex significantly, but does not attenuate optic nerve demyelination at 28 days post exposure
To investigate whether MCC950 could modulate neuroinflammation in the visual cortex, we initially employed immunofluorescence and Western blot to localize and quantify NLRP3 protein expression. Our findings revealed no statistically significant difference in NLRP3 expression between the BE 24 h group and the BE 24 h + MCC950 group (p > 0.05). However, compared to the BE 28 d group, MCC950 treatment significantly suppressed NLRP3 expression in the visual cortex (p < 0.05) (Fig 5A, 5B, 5E, 5F). Additionally, the co-localization ratio of NLRP3 with NeuN+ neurons was markedly reduced (p < 0.001) (Fig 5C). These findings suggest that MCC950 effectively inhibited the expression of inflammatory factors in the visual cortex at BE 28 d. Consistently, the expression level of cleaved Gasdermin D in the visual cortex was also significantly reduced in the BE 28 d + MCC950 group (p < 0.01) (Fig 5E, 5I). Our experimental results revealed a significant reduction in NeuN+ neuron and TUNEL co-localization (p < 0.05) (Fig 5H, 5J), accompanied by a notable increase in neuronal population (p < 0.05) (Fig 5K). These results collectively indicate that the NLRP3 inhibitor effectively modulates inflammatory responses and inhibits neuronal pyroptosis in the visual cortex at BE 28 d.
MCC950 Attenuates Neuroinflammation and Significantly Suppresses Pyroptosis, But Doesn’t Attenuate Optic Nerve Demyelination.(A) Representative images of immunofluorescence staining for NeuN (green), NLRP3 (red), and Iba-1 (grey) in mouse visual cortex slices, as well as group information, are shown in the images. The red triangle marks indicate the co-localization of NLRP3 and NeuN. The scale bars for all fluorescence intensity channels and the merge are set at 50 μm, the magnified inset is 20 μm, n = 4. (B) Quantitative analysis of visual cortex immunofluorescence density in NLRP3, n = 4. (C) Fluorescence intensity of NeuN+NLRP3+/NeuN+, n = 4. (D) Quantitative analysis of visual cortex immunofluorescence density in Iba-1, n = 4. (E, F, G, H) Western blot analysis of Iba-1, cleaved Gasdermin D and NLRP3 in visual cortex lysates. The molecular mass is indicated in kilodaltons, n = 3. (I, J) Quantitative analysis of representative fluorescence images of NeuN staining with TUNEL labeling in the peri-contusion region from different groups that information is shown in images, Scale bar = 100 μm, n = 4. (K) Quantitative analysis of visual cortex NeuN+ cells. (L) Representative images of immunofluorescence staining for MBP (green) and βIII-Tubulin (red) in mouse optic nerve slices. Group information is shown in images. Scale bar = 20 μm, n = 4. (M) optic nerve MBP density in different groups, n = 4. (N) optic nerve relative fluorescence intensity of MBP +/β III Tubulin + in different groups, n = 4. (O) TEM analysis of the optic nerve between the BE 24 h and BE 28 d groups, red stars indicate axons with obvious demyelination. Error bars indicate mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, ns p > 0.05.
To investigate whether MCC950 could attenuate optic nerve injury caused by blast exposure, we quantified the expression of MBP at 24 h and 28 d post BE (Fig 5L). The results showed no significant difference in the fluorescence of MBP/βIII tubulin in the optic nerve between the BE 24 h and BE 28 d groups (p > 0.05) (Fig 5M, 5N). Besides, TEM analysis revealed obvious demyelination in both the BE 24 h and BE 28 d groups and no significant alleviation with the administration of MCC950. Morphological observations showed widened axonal spaces and reduced axonal density in the BE 28 d group and the BE 28 d + MCC950 group within the same field of view (Fig 5O). These results indicate that MCC950 does not prevent optic nerve demyelination post BE.
Discussion
BTBI remains a subject of extensive research due to its complex injury mechanisms and subtle clinical manifestations, despite the varying designs of explosion injury models across laboratories [15,38,39]. The generated shock waves must simulate the Friedlander waveform. In our study, we utilized a biological shock tube (BST-I) that primarily simulates explosion shock wave overpressure by compressing air, enabling the simulation of blast-induced trauma injury in open or confined spaces and varying degrees of injury severity [26,40]. This makes our animal model more closely resemble the bTBI caused by real explosions and the validity of the model was verified through mNSS. Based on the mNSS scores of the bTBI animal model, the mTBI range was set at 0–6. Notably, an mNSS score of 0 does not imply that these mice were completely normal. Previous studies have indicated that in mTBI models, some mice do not exhibit overt neurological deficits [41]. This phenomenon likely reflects the reality of blast exposure in open-field conditions, where minor positional differences among the animals may lead to subtle variations in the intensity, angle, and frequency of the shock waves to which their heads are exposed, resulting in different degrees of injury outcomes.
We selected F-VEP, a non-invasive method for recording brain electrical activity, as the assessment tool to evaluate the neurophysiological function of the visual pathway [42,43]. Using F-VEP, we conducted assessments at multiple time points with a large sample size. Our findings revealed that visual function was significantly impaired 24 h post BE. After rapid recovery, visual function significantly decreased at 28 days post exposure. In addition, serial F-VEP recordings were performed on the same cohort of mice at predetermined intervals. This approach enabled precise one-to-one correspondence between animals and their respective electrophysiological data across all time points. Therefore, facilitating accurate assessment of individual progression and minimizing inter-animal variability. This longitudinal design enhances the reliability of our findings by controlling for individual differences and providing a comprehensive evaluation of temporal changes in visual function. In addition, leveraging interdisciplinary research encompassing ophthalmology and neurology [44,45], we conducted behavioral assessments using the light-dark box test to investigate visual dysfunction. At the two time points that F-VEP indicated delayed visual function, the behavioral tests revealed impaired light-dark discrimination in the experimental mice. These findings robustly corroborate our F-VEP results. These results systematically reveal the dynamic changes of visual function in mice after blast exposure. Meanwhile, this process of visual function reduction–recovery–further reduction suggests that the visual dysfunction associated with bTBI may involve distinct underlying pathological processes.
A large number of clinical studies and animal experiments have confirmed that visual evoked potentials originate from the activities of the visual cortex, and also represent the integrity of the visual pathway [46–48]. Previous studies have shown that most of the morphological changes after blast exposure usually occur in the eyeball and optic nerve, with retinal ganglion cells and optic nerve damaged [15,49–51]. We focused on two critical regions that represent the extracranial and intracranial segments of the visual pathway: the optic nerve and the primary visual cortex. The extent of latency delay strongly correlates with the degree of myelin loss in the optic nerve of animal models of demyelination [52]. Immunofluorescence assays revealed a significant reduction in MBP. As a critical structural component necessary for myelin formation, MBP serves as an objective biochemical marker for CNS damage and acute demyelination [53,54]. We found that optic nerve demyelination could be observed 24 hours post BE, and it continued to progress and was not affected by the inhibitors. Given that optic nerve injury can induce secondary degeneration in the visual cortex [9,55], we analyzed the changes in V1. Our investigation revealed a time-dependent increase in inflammatory markers in V1 from 24 hours to 28 days post BE. At 28 days, the expression of NLRP3 inflammasome was upregulated, triggering the expression of cleaved Gasedermin D and neuronal pyroptosis. These findings are particularly significant because previous studies have demonstrated that the maturation of cytokines IL-1β and IL-18 and the processing of the Gasdermin D, mediate cytokine release and induce pyroptosis [18]. In addition, by intervention with the specific inhibitor MCC950 of NLRP3, we significantly alleviated the blast–induced visual dysfunction at 28 days after exposure. Consistent with the findings of Evans et al. [49], we showed that specific inhibition of NLRP3 expression in the visual cortex using MCC950 significantly reduced neuronal pyroptosis, further supporting the role of inflammation in aggravating visual dysfunction. The visual pathway involves extensive cortical regions, and its function depends on precise neural circuits, which makes it particularly vulnerable to neural trauma [9]. Previous studies have confirmed that cell pyroptosis significantly affects neural function. We observed that neuronal pyroptosis occurred simultaneously with visual dysfunction [56]. Therefore, we hypothesized that blast wave could directly cause optic nerve demyelination and affect early visual function, which is consistent with previous clinical studies reporting the presence of significant VEP changes in patients with demyelinating optic nerve inflammation [57]. The temporal correlation between late visual dysfunction and pyroptosis of visual cortex neurons, may be a key factor in late visual dysfunction after blast exposure. These conclusions not only provide important supplementary evidence for the different pathological mechanisms of visual dysfunction after shock wave exposure, but also suggest that F-VEP detection plays a significant role in auxiliary assessment of bTBI. F-VEP, as a non-invasive and sensitive functional detection method, can objectively reflect the electrical activity of the visual cortex and the integrity of the visual pathway [58]. Importantly, the neurological deficits caused by shock wave exposure are relatively mild. By observing the changes in visual function, we will provide a new perspective for the auxiliary diagnosis and disease monitoring of bTBI.
Although optic nerve demyelination persisted throughout the observed pathological process, our findings demonstrated that MCC950 treatment effectively suppressed neuronal pyroptosis in the visual cortex and preserved visual function at 28 days post exposure. This suppression of pyroptosis may directly contribute to the preservation of visual function. As discussed in the review by McKenzie et al. [56], the inhibition of pyroptosis in central nervous system disorders can significantly enhance neurological function. Therefore, we hypothesize that the reduction in neuronal pyroptosis and inflammation may compensate for the effects of demyelination by preserving neuronal function, and enhancing visual conduction. In addition, we observed a gradual recovery of visual function between 3 days and 7 days post BE. Post traumatic edema in the nervous system has been widely reported [59–61]. We hypothesize that this phenomenon may be attributed to acute optic nerve edema induced by the extreme positive and prolonged negative pressure phases. As the condition transitions to the subacute phase, the resolution of edema may facilitate the gradual recovery of visual function. There is currently limited evidence specifically addressing optic nerve edema, which represents a crucial direction for our future investigation. Moreover, in recent years, peptides have been recognized as a rich source for drug discovery [62–64] and some peptides have shown promising therapeutic potential for brain injury [65–67], and the possible application of these peptides on visual injury induced by bTBI should be explored and discussed more deeply in the future.
Conclusion
In conclusion, bTBI presents challenges in terms of diagnosis and treatment because of its unique injury process and complex pathological mechanisms. Our research characterized the dynamic changes in visual dysfunction after blast exposure, and revealed that optic nerve demyelination and pyroptosis of visual cortex neurons are important mechanisms leading to visual dysfunction after exposure. Importantly, our findings suggest that early intervention in the formation of the NLRP3 inflammasome might be a potential intervention method to mitigate late visual dysfunction. In addition, through non-invasive visual electrophysiological examination, it provides a new perspective for the auxiliary assessment and monitoring of bTBI.
Supporting information
S1 FigbTBI model was induced in the BST-I apparatus in C57BL/6J. (A) Schematic representation of BST-I system. (B) Temporal changes in pressure during the instant of an explosion.(TIF)
S2 FigThe schedule of MCC950 treatment and its experimental analysis.The red arrows indicate the injection time points of MCC950, and the black arrows indicate the time points for experimental analysis.(TIF)
S3 FigQuantitative analysis of P2 latency(A) and N2-P2 amplitude (B) between the sham group and BE groups at 24 hours, 3 days, 7 days, 28 days,42days and 56 days.** p < 0.01, *** p < 0.001, ^ns^ p > 0.05.(TIF)
S4 FigMinimal data set The values used to build graphs.(ZIP)
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