
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

72318
10.1038/s41598-024-72318-x
Article
Roles of HMGB1 on life-threatening traumatic brain injury and sequential peripheral organ damage
Kawai Chihiro 1
Miyao Masashi miyaom@fp.med.kyoto-u.ac.jp

1
Kotani Hirokazu 2
Minami Hirozo 1
Abiru Hitoshi 1
Tamaki Keiji 1
Nishitani Yoko 1
1 https://ror.org/02kpeqv85 grid.258799.8 0000 0004 0372 2033 Department of Forensic Medicine, Kyoto University Graduate School of Medicine, Yoshida-Konoe-cho, Sakyoku, Kyoto, 606-8501 Japan
2 https://ror.org/01529vy56 grid.260026.0 0000 0004 0372 555X Department of Forensic Medicine and Sciences, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 Japan
13 9 2024
13 9 2024
2024
14 2142112 4 2024
5 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Traumatic brain injury (TBI) has been found to be associated with certain peripheral organ injuries; however, a few studies have explored the chronological influences of TBI on multiple organs and the systemic effects of therapeutic interventions. Particularly, high-mobility group box 1 (HMGB1) is a potential therapeutic target for TBI; however, its effects on peripheral organs remain unclear. Therefore, this study aimed to determine whether severe TBI can lead to multiple organ injury and how HMGB1 inhibition affects peripheral organs. This study used a weight drop-induced TBI mouse model and found that severe TBI can trigger short-lived systemic inflammation, in the lungs and liver, but not in the kidneys, regardless of the severity of the injury. TBI led to an increase in circulating HMGB1 and enhanced gene expressions of its receptors in every organ. Anti-HMGB1 antibody treatment reduced neuroinflammation but increased inflammation in peripheral organs. This study also found that HMGB1 inhibition appears to have a beneficial role in early neuroinflammation but could lead to detrimental effects on peripheral organs through decreased peripheral immune suppression. This study provides novel insights into the chronological changes in multiple organs due to TBI and the unique roles of HMGB1 between the brain and other organs.

Keywords

Traumatic brain injury
Weight drop model
Multiple organ injury
High mobility group box 1
Peripheral immune suppression
Subject terms

Pathogenesis
Trauma
Japan Society for the Promotion of Science21K17324 Kawai Chihiro issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Traumatic brain injury (TBI) is a life-threatening condition that can result in significant neurological impairments for survivors. TBI also affects systemic organs and contributes to multiple organ dysfunction1. In fact, approximately 89% of patients with severe TBI reportedly experience dysfunction of at least one non-neurological organ system2. After severe TBI, a cascade of autonomic and inflammatory mediators is released into the circulation, resulting in widespread organ effects and dysfunction, including the heart, lungs, liver, gut, musculoskeletal system, and immune system1,3. In addition, a previous study has revealed that peripheral alterations initiated by TBI can also exacerbate neurological consequences, and create vicious cycles4,5. Therefore, studying the influence of TBI on distant organs could contribute to the development of comprehensive treatment strategies for patients with head trauma, potentially improving their prognosis.

Previous studies have primarily focused on the consequences of experimental brain injury on a single organ at specific time points6–10. However, there have been limited investigations on the simultaneous and chronological effects of simulated TBI on multiple organs. The degree to which the consequences of TBI, including vulnerability and duration, differ between organs remains largely unexplored. To address this issue, we used the weight drop-induced severe TBI mouse model to elucidate the changes that occur in organs throughout the body following TBI. The weight drop model has a high mortality rate, implying life-threatening TBI, and we anticipated that the most severe head trauma would result in more obvious changes in peripheral organs. Furthermore, weight drop model involves brain injury resulting from direct impact on the head, and the injury mechanisms are similar to those observed in humans11,12. Additionally, this model does not require any pre-trauma manipulation, rendering it a faster, more straightforward, and cost effective method to implement12.

Damage-associated molecular patterns (DAMPs) may play a role in mediating the systemic organ response that arises from local brain injuries caused by TBI. DAMPs are intracellular components that are released by damaged or dying cells and bind to pattern recognition receptors, such as TLR2, 4, 9, and Rage13. In cases of severe TBI, polytrauma, and diseases, DAMPs initiate an innate immune response, which can worsen the disease progression14–16. DAMPs extend beyond the local area and can have systemic effects when they enter the bloodstream17,18. High-mobility group box 1 (HMGB1) is a representative DAMP19 that is released from injured neurons and glial cells, and it has been implicated in inflammation and cell death in the surrounding tissues20,21. Therefore, HMGB1 is an attractive therapeutic target during the treatment of TBI22,23 although its roles in the blood of patients with TBI remain controversial. Some studies have suggested that HMGB1 acts as an inflammatory mediator in the lungs after TBI24, whereas others have suggested that plays a role in the development of central nervous system (CNS) injury-induced immune suppression and possible anti-inflammatory effects25. Thus, a comprehensive understanding of the effects of HMGB1 therapeutic interventions on multiple organs is necessary for developing effective treatments for TBI.

In this study, we comprehensively and chronologically examined the histological and functional changes that occur in the brain, lungs, liver, kidneys, and other organs of C57BL/6 mice that sustain clinically life-threatening TBI. We aimed to elucidate the systemic effects of the most severe forms of TBI. Furthermore, we investigated the potential of an anti-HMGB1 antibody for TBI in multiple organs of mice.

Results

Weight drop-induced head trauma model reflects clinically life-threatening traumatic brain injury

To validate the weight drop-induced head trauma model and assess the severity of TBI, we conducted survival and macroscopic analyses of mice subjected to weight drop-induced head trauma with falling heights of 5, 7.5, and 10 cm. All mice with a 10 cm weight falling height and 75% of those with a 7.5 cm weight falling height died, whereas the sham mice and 91.6% of those with a 5 cm weight falling height survived for 2 weeks. Some mice died immediately after the head trauma, whereas others died 3–5 days after the injury (Fig. 1a). Macroscopically, all mice exhibited damage to the right cerebrum. The 7.5 cm weight falling height caused contusion, subarachnoid hemorrhage, and increased vascular permeability, which were visualized and analyzed using the Evans blue dye pre-injection (Fig. 1b). In contrast, the 5 cm weight falling height only resulted in a slight subarachnoid hemorrhage, and no extravasation of Evans blue dye was detected. Quantification analysis of the extravasation of Evans blue dye showed that vascular permeability exhibited an immediate increase, at 6 h, in the damaged right hemisphere, and worsened over the next 3 days (Fig. 1c). Furthermore, the extravasation occurred in the intact left hemisphere. The mice subjected to the 7.5 cm weight falling height exhibited severe neurological impairment, with their Neurological Severity Score (NSS) having a mean of 6.0 ± 1.8 at 1 day post-trauma. However, they immediately began to recover and their mean NSS recovered to 1.0 ± 1.0 as long as they survived (two out of the five mice died at 7 days post-trauma). Mice exposed to a 5 cm weight falling height showed slight neurological impairment (Fig. 1d). These results suggest that the weight-drop-induced head trauma model reflects varying degrees of TBI, ranging from mild (5 cm) to life-threatening (7.5 cm) and even fatal (10 cm). Furthermore, the fact that some mice that survived the initial phase of life-threatening TBI and displayed signs of neurological recovery nevertheless died 1–7 days after TBI suggests the impact of secondary injuries beyond the brain.Fig. 1 Validation and severity assessment of weight drop-induced TBI. (a) Survival rates of sham and TBI mice depending on falling height. The figure depicts the number of mice that were operated on and the number of surviving mice at each designated time point. (b) Macroscopic images of the brains from sham and TBI (5 and 7.5 cm weight falling height) mice at 24 h post-trauma. Arrows indicate the points of impact. Yellow arrowheads indicate subarachnoid hemorrhage. Red arrowheads indicate extravasation of Evans blue dye. The scale bars represent 1 cm. (c) Evaluation of the increase in cerebral vascular permeability in sham and TBI (7.5 cm falling height) mice.at 6, 24, and 72 h post-trauma. Vascular permeability was evaluated by measuring the extravasation of Evans blue dye. Data are presented as the means ± SEM. Statistical analyses were performed using Student’s t-test. **P < 0.01. n ≥ 5 for all groups. (d) Neurological Severity Score (NSS) measured over 7 days in TBI (5 cm and 7.5 cm weight falling height) mice or sham-operated mice. The number of surviving mice at each designated time point is indicated in the figure. Data are presented as the means ± SD. Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test. †P < 0.05, ††P < 0.01 vs. 5 cm falling height TBI mice and sham operated mice.

Chronological changes in the brain with life-threatening TBI: rapid temporal inflammation and delayed glial activation

To assess chronological changes in the brain following severe TBI, histological and immunohistochemical analyses were conducted on the right hemisphere near the impact point, where the weight dropped from 7.5 cm falling height, and on the left intact hemisphere (Fig. 2a,b). The mortality rates and the sampling numbers in each analysis are shown in Supplementary Fig. S1. At 6 h post-trauma, a remarkable increase was observed in the swelling of the edematous right cortex, and this edema peaked at 24 h. However, no significant changes were noted at 6–72 h. These results were consistent with those of the Evans blue dye analysis (Fig. 1c), indicating similar chronological changes. Additionally, the influx of neutrophils into the right cortex, particularly in areas of bleeding and contusion, significantly increased 6 h post-trauma. In the right lateral ventricle, located beneath the site of cerebral contusion, an increase in neutrophils with hemorrhage was observed immediately following TBI, particularly near the choroid plexus (Supplementary Fig. S2). Astrocytes stained with glial fibrillary acidic protein (GFAP) slightly extended the processes at 24 h post-trauma, and significantly increased and activated hypertrophic gliosis at 72 h. Simultaneously, the activation of ionized calcium-binding adapter molecule 1 (Iba-1) positive microglia was observed. Microglia showed a hypertrophic or bushy morphology at 24 h, and amoeboid microglia significantly increased at 72 h.Fig. 2 Chronological changes in brains with severe traumatic injury. (a, b) Histopathological and immunohistochemical analyses of the brains with life-threatening TBI (7.5 cm weight falling height) and controls. (a) Brain sections were stained with Klüver-Barrera (KB) and hematoxylin and eosin (H&E) and immunostained with the neutrophil marker lymphocyte antigen 6G (Ly6G), the activated astrocyte marker; Glial fibrillary acidic protein (GFAP), and the microglia marker; ionized calcium-binding adapter molecule 1 (Iba-1). Whole images of KB-stained sections, high-magnification images of H&E-stained sections, and immunostained sections of the boxed areas are shown. Scale bars represent 1 mm (whole image) and 100 µm (high magnification), respectively. CTX, cerebral cortex; CC, corpus callosum. (b) Relative thickness of the cerebral cortex was calculated using the ratio of the average cortex thickness from left to right in five randomly chosen fields from each section. The number of Ly6G positive cells, astrocytes, and microglia were counted in 10 randomly chosen fields from each right and left hemisphere and converted per mm2. GFAP and Iba-1 positive areas were measured in 10 randomly chosen fields. (c) Quantitative RT-PCR analysis of the right cerebrum in control and TBI mice at the indicated time points. Gene expressions are normalized to Gapdh expression. (d) Immunohistochemical analyses of the progression of GFAP-positive regions in the hippocampal formation. Damaged and necrotic areas are highlighted with dotted lines. High-magnification images of the boxed areas show the affected hypothalamus. Scale bars represent 1 mm (low magnification) and 100 µm (high magnification). HPF, hippocampal formation; HY, hypothalamus; TH, Thalamus. (e) Histological evaluation of hypothalamic gliosis in control and TBI mice. (f) Serum norepinephrine levels in control and TBI mice. Data are presented as the means ± SEM. Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01. n ≥ 5 for all groups.

To gain a more comprehensive understanding of the chronological alterations in the brain following severe TBI, quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis was conducted on gene expressions in the damaged right cerebrum (Fig. 2c). The mRNA expression levels of cytokines and chemokines rapidly increased at 6 h, and most of them gradually decreased at 24 h. Meanwhile, the levels of anti-inflammatory cytokine (Tgf-β) and innate immune receptors, related to DAMPs (Tlr2, 4, 9, and Rage), were either increased or maintained at high levels for 72 h. The expression levels of the neutrophil chemokine receptor (Cxcr2) and macrophage marker (F4/80) correlated well with the histological changes observed in neutrophils and microglia (Fig. 2b). The aquaporin-4 (Aqp4) expression levels, a key water channel protein in the CNS that plays a role in causing cerebral edema26, increased significantly at 72 h post-treatment (Fig. 2c). No noticeable histological or functional alterations were observed in the sham mice after 24 h (Supplementary Fig. S3). Moreover, rapid progression of gliosis in the hypothalamus, which regulates the autonomic nervous system, was observed in the damaged right hemisphere 72 h post-trauma (Fig. 2d,e), where no severe damage, necrosis, neutrophil infiltration, or microglial activation was detected (Supplementary Fig. S4). This was accompanied by a change in serum norepinephrine levels (Fig. 2f). These results suggest that even severe TBI, which is life-threatening, triggers an acute inflammatory response that subsequently subsides in the subacute phase despite the ongoing progression of gliosis and microglial activation. In contrast, the weight-drop-induced gliosis developed in remote locations, such as the hypothalamus, may lead to unforeseen disruptions of the autonomic nervous system.

The impact of life-threatening TBI across multiple organs characterized by transient changes during the acute phase, with varying susceptibility and progression

To clarify chronological changes in multiple remote organs following fatal TBI, histological and functional analyses of the lungs, liver, and kidneys were performed. The lungs showed immediate pulmonary impairment, lung congestion and neutrophil infiltration in the interstitial region, which peaked at 6 h post-trauma and subsided with time (Fig. 3a,b). The analysis of bronchoalveolar lavage (BAL) fluid did not reveal surge in neutrophils or lymphocytes within the alveolar space. (Supplementary Fig. S5a). The mRNA expressions of inflammatory cytokines, chemokines, and chemokine receptors in the lungs also significantly increased only at 6 h (Fig. 3d). In contrast to the transient deterioration observed in the lungs, the liver exhibited a bimodal response to TBI. Histological examination revealed only ephemeral neutrophil infiltration in the liver (Fig. 3e,f); however, the serum levels of hepatic injury markers persisted until 72 h later (Fig. 3g, Supplementary Fig. S5b). The mRNA expressions of the neutrophil chemokine receptor (Cxcr2) and neutrophil-related chemokines (Il-8) were elevated only 6 h post-trauma. Additionally, qRT-PCR analysis revealed an unforeseen increase in mRNA expressions of macrophage-related chemokines (Cxcl2, Ccl2, and Ccl3), and inflammatory cytokines (Tnf-a) at 72 h (Fig. 3i). In the kidney, TBI caused no significant renal injury, except for slight temporal neutrophil infiltration, and no notable changes were found in biochemical and functional analysis (Fig. 3j–n, Supplementary Fig. S5c). The sham mice exhibited a slight increase in neutrophil infiltration in the lungs and liver at 6 h, although this increase was considerably less than that in TBI mice (Supplementary Fig. S6a-k). Interestingly, in all three organs, including the kidney, which experienced the least alteration, comparable increases were observed in the mRNA expression of Rage, a receptor for HMGB1, at 6 h post-trauma (Fig. 3d,i,n). These results suggest that HMGB1 is released into the circulation following TBI and exerts an influence on multiple organs. Inflammatory changes were observed only at 6 h post-trauma, and no evidence of neutrophil reinfiltration was found at 72 h. However, histological examinations conducted at 72 h revealed interstitial edema in the lungs and liver, characterized by thickening of the alveolar walls or hepatic sinusoids (Fig. 3a,e). To quantify the changes in vascular permeability on multiple organs, including the lungs, heart, liver, spleen, and kidneys, post-traumatic extravasation analyses were performed based on the Evans blue dye. At 6 h, when neutrophil infiltration was most remarkable, no alteration of vascular permeability was found (Fig. 3c,h,m, Supplementary Fig. S5d); however, at 72 h, increased vascular permeability was observed in every organ, with the liver and spleen being the most conspicuous. It has been suggested that the increased vascular permeability on multiple organs could be attributed to either inflammation or autonomic dysregulation3,27. However, in this study, the serum levels of pro-inflammatory cytokines, tumor necrosis factor (TNF) -α and interleukin (IL)-1β did not increase in mice at 72 h post-trauma (Supplementary Fig. S7). These findings indicate that life-threatening TBI triggers rapid neutrophil activation throughout the body. Despite its potentially fatal consequences, the inflammatory response is short-lived and has minimal impact on multiple organ functions. Therefore, DAMPs may mediate these acute phase changes. Additionally, in the subacute phase, autonomic dysfunction, such as neurocirculatory failure, may be more critical for multiple organs.Fig. 3 Chronological evaluation of changes in the lungs, liver, and kidneys in control mice and those with severe TBI (7.5 cm weight falling height) at 6, 24, and 72 h post-trauma. (a–d) Histopathological and functional lung evaluations of control and TBI mice. (a) Hematoxylin and eosin (H&E) staining and immunostaining with lymphocyte antigen 6G (Ly6G); yellow arrowheads, neutrophil infiltration; red arrowheads, Ly6G + cells. (b) Histological counts of Ly6G + cells, and histological lung injury scores. (c) Evaluation of the changes in vascular permeability following either TBI or sham operation. (d) Quantitative RT-PCR analysis of the lungs. Gene expressions are normalized to Gapdh expression. (e–i) Histopathological and functional liver evaluations. (e) H&E staining and immunostaining with Ly6G. PV, portal vein. (f) Histological counts of Ly6G + cells. (g) Serum levels of alanine transaminase (ALT). (h) Evaluation of the changes in vascular permeability. (i) Quantitative RT-PCR analysis of liver. (j–n) Histopathological and functional kidney evaluation. (j) H&E staining and Ly6G immunostaining. G, glomeruli. (k) Histological counts of Ly6G positive cells. (l) Serum levels of blood urea nitrogen (BUN). (m) Evaluation of the changes in vascular permeability. (n) Quantitative RT-PCR analysis of the kidneys. Scale bars, 50 µm. Data represent the means ± SEM. Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test for multiple comparisons or Student’s t-test. *P < 0.05, **P < 0.01. ††P < 0.01 vs. sham. NS, not significant. n ≥ 5 for all groups.

Circulating HMGB1 following life-threatening TBI could cause unique effects on the brain and other organs

Examination of peripheral organs over time revealed that the expression of Rage mRNA exhibited similar chronological changes over time (Fig. 3). HMGB1, a ligand for RAGE, initiates a systemic response and contributes to the progression of multiple organ failure. Therefore, to evaluate the release and participation of HMGB1 in TBI, we performed an immunohistochemical analysis of HMGB1 translocation. In the injured cerebral cortex near the necrotic area, HMGB1 was released from the nucleus at 6 h post-trauma and was partially lost at 24 h post-trauma (Fig. 4a). Additionally, we found that serum HMGB1 levels in mice with severe TBI were significantly elevated until 6 h after trauma, as evidenced by enzyme-linked immunosorbent assay (ELISA) results (Fig. 4b). As immunohistological changes were only observed in the brains (Supplementary Fig. S8), circulating HMGB1 could, at least partially, have been released from the injured cortex. Extracellular HMGB1 plays a significant role in escalating neuroinflammation following TBI, and the blockade of HMGB1 can diminish cerebral edema, blood–brain barrier (BBB) disruption and inflammatory reactions22. To assess the potential effects of HMGB1 on other organs following TBI, we next administered an anti-HMGB1 neutralizing antibody to mice before and after TBI induction. As shown in the results, it is noteworthy that antibody treatment caused a reduction in brain inflammation but concurrently led to an aggravation of lung and liver inflammation (Fig. 4c). In every organ, the expressions of mRNA for Rage were significantly decreased by anti-HMGB1 antibody treatment, implying that the HMGB1 blockade was sufficiently achieved. Histological examination also revealed that anti-HMGB1 neutralizing antibody promoted pulmonary interstitial neutrophils and liver sinusoidal neutrophils following TBI (Fig. 4d). BAL fluid analysis and biochemical analysis revealed no differences between the groups treated with the anti-HMGB1 antibody and the isotype-treated controls (Fig. 4e, Supplementary Fig. S9). The differences observed between the brain and other organs can be attributed to the macrophage phenotype. These organs possess an abundance of resident macrophages that play critical roles in regulating innate immune responses through their pro-inflammatory and anti-inflammatory phenotypes28. The anti-HMGB1 neutralizing antibody tended to impede the migration of myeloid-derived suppressor cells (MDSCs) (Fig. 4f,g), which are typically characterized as CD11b+Ly6G/C+ cells29. The reduction in MDSCs, which regulate the immune system, led to a relative increase in the population of CD80+ pro-inflammatory macrophages compared to that of CD163+ anti-inflammatory macrophages in the lungs and liver (Fig. 4h). These results indicate that HMGB1, which is triggered by TBI, may be a crucial factor in regulating immune responses in the periphery and preventing secondary organ injury following TBI.Fig. 4 Role of high-mobility group box 1 (HMGB1) in brain and other organs following TBI. (a) HMGB1 immunostaining in the brains of controls and mice with TBI (7.5 cm weight falling height) at 6 and 24 h post-administration. Arrowheads indicate negative nuclei, indicative of HMGB1 translocation to the cytoplasm or extracellular space. Scale bars, 50 µm. (b) Serum levels of HMGB1 in controls and TBI mice at the indicated time points. Data are presented as the means ± SEM. Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01. (c) Quantitative RT-PCR analysis was conducted on the brains, lungs, and liver of mice with TBI that were administered intravenous injections of either anti-HMGB1 antibody or isotype control antibody 30 min before and 1 h post-trauma. (d) Ly6G immunostaining and histological analysis of Ly6G-positive cells in the lungs and liver. Scale bars, 50 µm. (e) Alanine transaminase (ALT) and creatinine (Cre) serum levels. (f) Representative images of co-immunofluorescence staining of Ly-6G/C and CD11b in the liver of TBI mice treated with anti-HMGB1 or isotype control antibodies. The image depicts the cells stained with antibodies against Ly-6G/C (red), CD11b (green), and DAPI (blue). Arrowheads indicate positive cells, and a red arrow indicates the presence of Ly-6G/C and CD11b co-positive cell. Scale bars, 20 µm. (g) Evaluation of the presence of cells co-expressing Ly-6G/C and CD11b by histological examination. The ratio of Ly-6G/C and CD11b co-positive cells to Ly-6G/C-positive cells was calculated for each liver tissue sample. Positive cells were counted in 10 randomly selected fields and their sum was determined. (h) Quantitative RT-PCR analysis of the lungs and liver. Data are presented as the means ± SEM. Statistical analyses were performed using Student’s t-test. *P < 0.05, **P < 0.01. NS, not significant. n ≥ 3 for all groups.

Discussion

In this study, we successfully replicated a life-threatening mouse model of TBI that closely mimics severe human head trauma. Despite the severity of the TBI, the inflammatory response triggered in multiple remote organs was short-lived and had minimal impact on the functioning of these organs. HMGB1 is released into the circulation in the early stages of TBI and may impact both the brain and peripheral organs. The use of anti-HMGB1 antibody therapy reduced neuroinflammation; however, it exacerbated peripheral inflammation.

The results of our life-threatening TBI model were consistent with previously reported clinical progression of the brain30,31, excluding the mortality rate. Notably, in our model, approximately half of the mice died 5 days post-trauma, even though their neuroinflammation subsided and their motor and neurobehavioral functions had been restored. This delayed death may have been related to the disruptions in the autonomic nervous system during the recovery period. Astrocytes play a crucial role in regulating neurological function; however, their excessive activation can lead to disruptions in normal function32. In this study, a decrease in blood norepinephrine was observed 3 days after the injury, which may have been indirectly promoted by the gliosis in the hypothalamus because the hypothalamus is the center of the autonomic nervous system regulation. However, we did not investigate the morphological and functional abnormalities of the heart that could be directly linked to death caused by autonomic nervous dysfunction. Therefore, it is crucial to conduct additional research to explore the underlying mechanism of delayed death. This should involve conducting longitudinal measurements of modifications in brain function and organ changes, and comparing the findings with prognosis.

In our model of life-threatening TBI, despite the severity of the TBI, only transient inflammation was observed in some organs with minimal impact on their functioning. The influence of TBI on multiple organs differed during the initial response. Severe polytrauma, which leads to hypovolemia and hypoxia, triggers the activation of the entire body’s defense system33, including the recruitment of neutrophils27. TBI also supposedly has widespread effects on the body through autonomic dysregulation and systemic inflammatory responses, leading to multiple organ failure1,3. In this study, inflammatory responses, including neutrophil infiltration and cytokine expression, subsided after 6 h, which is the peak time of neuroinflammation following life-threatening TBI. TBI triggers rapid neutrophil activation throughout the body; however, despite its potentially fatal consequences, the systemic inflammatory response triggered by TBI is short-lived and has a lesser impact on multiple organ functions than previously thought. Additionally, in the kidneys, only slight intravascular increases in neutrophils were observed, and no elevation in cytokines or other factors was found. This implies that the kidneys exhibit a notable capacity for resilience following severe TBI, which is consistent with the notion that they are less vulnerable to early damage than the lungs and liver17,34. However, even mild TBI, such as a concussion, can contribute to pulmonary priming and promote extensive neutrophil accumulation in the interstitial space of the lungs, exacerbating secondary lung injury following head trauma7. Our study also showed a general trend of transient neutrophil elevation. Notably, even in cases of severe head trauma, there was only a slight increase in interstitial neutrophils, without an increase in alveolar space inflammatory cells. TBI, regardless of its severity, could primarily activate intravascular and intrastromal neutrophils, and then contribute to the priming of multiple organs and exacerbate secondary injuries triggered by other injuries, similar to the mechanism observed in the lungs.

While the consequences of neuroinflammation resulting from brain injury have typically been observed as acute and temporary, the disruption of neuromodulatory mechanisms associated with brain injury might have the potential to impact all organs, even during the recovery process. At 3 days post-trauma, when it was assumed that autonomic dysregulation had occurred but no reactivation of neuroinflammation was observed, minor increases were detected in liver injury markers and some chemokines without neutrophil reinfiltration. Additionally, increased vascular permeability was observed in all organs on the same day. During severe trauma, a sudden surge in catecholamines due to trauma-induced stimulation of the α-adrenergic system results in intense vasoconstriction, blood redistribution, and impaired endothelial cells of the capillaries, leading to interstitial edema27. However, in this study, levels of serum norepinephrine, a typical catecholamine, were found to decrease 3 days post-trauma. One possible explanation for this is that the observed increased vascular permeability may result from the transient extreme rise in catecholamines between 1 and 3 days post-trauma. Another possibility is that autonomic dysfunction leads to increased vascular permeability. Catecholamines play a role in ameliorating edema and inflammatory reactions35, and a lack of catecholamines may indirectly contribute to edema formation. To comprehend the pathophysiological processes that arise from the consequences of severe TBI on multiple organ systems, it is essential to simultaneously assess the chronological changes in neurological and organ function.

Circulating HMGB1 derived from injured brains may extend beyond the damaged brain tissue to act as a mediator in multiple peripheral organs. HMGB1 is a chromatin protein that not only arranges DNA and regulates transcription but also influences various biological processes depending on its subcellular or extracellular location19. Extracellular HMGB1 secreted by immune cells or released from damaged cells plays a role in blending sterile and infectious inflammatory responses following severe trauma or disease through multiple surface receptors, including TLR2, TLR4, and RAGE19. Moreover, in TBI, HMGB1 released from damaged neurons, glial cells, and inflammatory cells contributes to neuronal death, neuroinflammation, and BBB disruption20,21. In this study, the rapid release of HMGB1 into the circulation was observed. Furthermore, the upregulation of mRNA expression of Rage in multiple organs, including the kidney, was noted at 6 h post-trauma, indicating that circulating HMGB1 partially affected multiple peripheral organs in the early stage of TBI. Based on these findings, we propose that HMGB1 functions as an early mediator of peripheral inflammation, particularly at 6 h post-TBI and initiates neutrophil infiltration into multiple organs.

The release of HMGB1 induced by TBI appears to function primarily as an anti-inflammatory agent in peripheral tissues, contrary to the hypothesis that it acts as a pro-inflammatory mediator. HMGB1 is released from damaged brain tissue, exacerbating cerebral damage36, and anti-HMGB1 antibody therapy not only mitigates these harmful effects22 but also facilitates the recovery of neurological function in the later stages of TBI23. Therefore, targeting HMGB1 is a promising therapeutic option for TBI. However, to the best of our knowledge, only a few studies have explored the role of anti-HMGB1 antibodies in other organs following TBI37. Thus, we administered an anti-HMGB1 antibody both before and after TBI and examined its therapeutic effects on the lungs and liver. As expected, inflammation was attenuated in the brain, and the mRNA expression of Rage reduced in all organs because of the blocked ligand. Surprisingly, inflammation exacerbated in the liver and lungs, with increased expressions of cytokines and neutrophils. These results imply that HMGB1 plays more complex roles in TBI, which differ in the brain and peripheral organs. The release of HMGB1 due to TBI, especially life-threatening TBI, appears to primarily serve an anti-inflammatory function in peripheral tissues rather than promoting inflammation.

The release of HMGB1 from injured brains may contribute to the suppression of peripheral immunity by promoting the migration of MDSCs. The suppression of peripheral immunity has been reported to be a conflicting physiological response to acute brain lesions38. This phenomenon is associated with increased susceptibility to nosocomial infections, such as pneumonia, following TBI, which is sometimes referred to as CNS-induced immune deficiency syndrome39. It has been suggested that the brain immune signaling mechanism serves to prevent excessive inflammation in the CNS from spreading to the periphery38,40, although the underlying mechanisms remain poorly understood. One possible cause is the proliferation and accumulation of circulating MDSCs29, which regulate peripheral immunity by modulating macrophage activity. Specifically, they activate anti-inflammatory M2 macrophages41,42, which have been shown to have a protective effect in various inflammatory conditions. Additionally, animal studies have indicated that HMGB1 is involved in immune suppression following CNS injury. HMGB1 has been linked to the expansion of MDSCs in vivo and to promote the infiltration of MDSCs into peripheral tissues following experimental brain ischemia25. In this study, the administration of anti-HMGB1 antibodies resulted in a reduced infiltration of MDSCs into the liver tissue following TBI. Furthermore, the administration of anti-HMGB1 antibodies enhanced the inflammatory M1 population and decreased the percentage of the anti-inflammatory M2 population in both the liver and lungs. These findings suggest that HMGB1 plays a crucial role in regulating peripheral immune responses in multiple organs after TBI by activating MDSCs. We employed a life-threatening TBI model, which may have resulted in a more pronounced inhibition of peripheral inflammation by MDSCs, thereby making organ damage less noticeable. Therefore, excessive release of HMGB1 exacerbates neuroinflammation in the brain, which is consistent with previous research that focused on targeting this protein as a therapeutic target in head trauma. However, HMGB1 inhibition may have unintended consequences for distant organs by removing the brakes during CNS injury-induced peripheral immunosuppression, which could result in multiple organ failure. This study highlights the importance of considering the effects of anti-HMGB1 therapy not only on the brain but also on the entire body.

This study had some limitations. We aimed to use life-threatening TBI as a means of better understanding the changes that occur in various organs due to head trauma. However, it is important to note that a quarter of the mice in our study were destined to survive, while the others were destined to die. This survival bias may have led to the selection of relatively minor injuries among the severely injured mice, especially at 72 h. To address this, it is necessary to compare with mice that have suffered brain injuries in other TBI models, such as controlled cortical impact and fluid percussion models, which create a more uniform brain injury11. Additionally, although we have proposed the potential involvement of the HMGB1-related pathway in MDSCs migration and activation following TBI, we have not fully evaluated this mechanism. Because administration of anti-HMGB1 antibodies in the current study merely inhibited circulating HMGB1. Furthermore, it is important to note that the administering the anti-HMGB1 antibody before TBI does not reflect a typical clinical situation. We have not ruled out the possibility that administering the HMGB1 antibody sensitizes peripheral organs and exacerbates peripheral organ injury following TBI, rather than HMGB1 contributing to the suppression of inflammation in peripheral organs through a complex pathway. Therefore, future research using conditional knockout of HMGB143, particularly in astrocyte-specific knockout mice44, is necessary to fully elucidate the influence of HMGB1 on peripheral organ changes following TBI.

In conclusion, we successfully replicated a life-threatening mouse model of TBI that closely mimics severe human head trauma. The most significant impact of brain injury on various organs is the inflammatory response in the acute phase. However, despite the severity of the TBI, the inflammatory response triggered in remote organs was found to be short-lived and had a minimal impact on the functioning of these organs. Additionally, this study indicates that the release of HMGB1 following TBI could have different effects on the brain and peripheral organs. The use of HMGB1-targeted therapy for TBI is expected to reduce neuroinflammation and improve survival; however, it may also have unintended consequences such as exacerbating damage to peripheral organs. This study provides novel insights into the chronological changes in multiple organs following TBI, and the unique roles of HMGB1 between the brain and other organs. To develop effective and safer treatments, careful evaluation of the effects of therapeutic agents on the entire body is essential.

Methods

Animal models

In this study, male C57BL/6 mice (20–25 g, 8–10 weeks old; Japan Inc., Tokyo, Japan) were used. The mice were under specific pathogen-free conditions in an air-conditioned room at 23 °C with a 12-h light/dark cycle. They had access to food and water ad libitum. To create an experimental TBI model, we utilized a weight-drop-induced TBI model12,45. First, the animals were administered general surgical anesthesia with an intraperitoneal injection of a combination anesthetic (0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol)46 and their heads were secured in place using a clamp. The fur was shaved, and a midline longitudinal incision was made to expose the skull. A Teflon-tipped cone (2 mm in diameter) was placed 1–2 mm lateral (right) to the midline in the mid-coronal plane, and a 100 g weight was dropped on the cone from different heights, resulting in a focal injury to the right hemisphere. We made modifications to this model by equipping the cones with stoppers, which ensured that the cone tips would not penetrate the head by more than 2 mm. Subsequently, the skin was then sewn back together and the animals were taken out of the clamp. The sham-operated controls were those that underwent only an incision and were not subjected to the weight-drop trauma. Subjects in the healthy control group received anesthesia without undergoing any surgical procedures.

For survival analysis, mice were subjected to varying degrees of brain injury using a weight-drop device with drop heights of 5, 7.5, and 10 cm, or they were underwent a sham operation. The animals were monitored for 2 weeks for any signs of distress, and those exhibiting severe distress, such as difficulty breathing, bluish skin color, or lack of response to touch, were euthanized by cervical dislocation. These mice were recorded as having experienced TBI-induced mortality. To conduct a time-course analysis, mice were subjected to TBI using a 7.5-cm weight falling height and were sacrificed at 6, 24, and 72 h post-trauma to collect samples for further analysis. Healthy control mice were only administered surgical anesthesia and sacrificed at 6 h after anesthesia. Additionally, the sham mice were sacrificed 6 and 24 h after the sham operation. To determine the potential role of HMGB1 in the brain and other organs following life-threatening TBI, the mice were intravenously administered an anti-HMGB1 antibody (SHINO-TEST, Tokyo, Japan) at a dosage of 2 mg/kg 30 min before and 1 h after TBI, with a falling weight of 7.5 cm. The mice were monitored and sacrificed 6 h after TBI. A vehicle control group of mice was injected with the control IgY fraction (Shino-Test) and subjected to TBI.

All experimental protocols were approved by the Animal Care and Use Committee of Kyoto University Graduate School of Medicine (Med Kyo 21,064), and were performed in accordance with the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences. This study is reported in accordance with the ARRIVE guidelines 2.047.

Assessment of neurological deficits

The NSS12 was used to evaluate neurological and cognitive impairments in mice that survived TBI from drop heights of 5 or 7.5 cm and sham-operated mice. The mice were tested at 1, 3, and 7 days post-trauma, and the score was determined by assessing 10 individual clinical parameters such as motor function, alertness, and physiological behavior. In the scoring system, one point was given for the absence or inability to perform a tested reflex or task, while no points were given for a successful performance. The detailed methodology and assessment criteria are provided as supplementary information (Supplementary Table S1). A maximum NSS (10 points) means severe neurological dysfunction characterized by the failure of all tasks (worst score).

Vascular permeability assays

Evans blue dye was used to investigate changes in vascular permeability in the brain, lungs, liver, heart, and spleen following TBI48. Mice were intravenously administered Evans blue (40 mg/kg; Sigma-Aldrich) 1 h before the TBI or sham operation. At indicated time points, each organ was harvested by perfusing with phosphate-buffered saline (PBS) for 1 min, followed by drying at 60 °C for 48 h. The Evans blue was extracted by immersion in 5 mL formamide at 37 °C for 24 h. The concentration of Evans blue in the supernatant was measured by absorbance at 620 nm using a spectrophotometer (GENESYS 10S UV–Vis, Thermo Scientific, Waltham, MA, USA) and calculated as μg/mg of dry tissue weight. The control groups were sacrificed and harvested 1 h after Evans blue administration.

Serum biochemical analyses

Blood samples were collected via cardiac puncture. Serum aspartate transaminase, alanine transaminase, blood urea nitrogen, and creatinine levels were analyzed by a Hitachi 7180 analyzer (Hitachi, Tokyo, Japan). Serum levels of IL-1β, TNF-α, norepinephrine, and HMGB1 were quantified using ELISA kits purchased from R&D Systems (Minneapolis, MN, USA), Abcam (Cambridge, MA, USA), or SHINO-TEST.

Histopathology

For light microscopy analyses, the mice were subjected to transcardiac perfusion with 4% paraformaldehyde, followed by harvesting of the brain, lungs, liver, and kidneys. Formalin-fixed organs were embedded in paraffin or an optimal temperature compound for frozen sections. Paraffin-embedded tissue sections of 4-μm thickness were cut and stained with hematoxylin and eosin and/or Klüver–Barrera. Cerebral edema was evaluated by measuring the thickness of the damaged right cortex relative to that of the intact left cortex27, with the average thickness being calculated from five randomly selected areas in each specimen. Lung injuries were scored according to the standardized guidelines set by the American Thoracic Society49. Quantification was performed on 10 randomly chosen areas at 400 × magnification.

Immunohistochemistry and immunofluorescent analyses

Immunohistochemical staining of paraffin-embedded specimens was performed in accordance with previously established protocols17. Heat induced antigen retrieval was performed by boiling in Tris–EDTA Buffer (pH 8.0) or 10 mM citrate buffer (pH 6.0). The slides were incubated overnight at 4 °C with the following optimally diluted primary antibodies: GFAP (rabbit, 1:400; Abcam), Iba1 (rabbit, 1:2000; Abcam), lymphocyte antigen 6G (rabbit, Ly-6G; 1:2000; Abcam), HMGB1 (rabbit, 1:1000; Abcam), or a negative control reagent. Following incubation with an anti-rabbit secondary antibody (goat polyclonal; pre-diluted; Medical & Biological Laboratories Co., Ltd., Nagoya, Japan), antibody binding was detected using the labeled polymer method. Diaminobenzidine was used as the chromogen. For the quantitative assessment, the staining of each specimen was captured in 10 randomly chosen areas in each specimen at 400 × magnification, and the numbers and areas of stained cells were measured using the ImageJ software. The count of positive cells in the brain was calculated per 1 mm2.

Immunofluorescence staining was performed on the formalin-fixed frozen tissue sections. The primary antibodies employed were lymphocyte antigen 6G/C (Ly-6G/C; rabbit; 1:1000; Abcam) and cluster of differentiation 11b (rat; 1:400; Abcam). Subsequently, the slides were then incubated with Alexa Fluor 488 goat anti-rabbit secondary antibody (Cell Signaling Technology, Beverly, MA, USA) and Alexa Fluor 594 phalloidin. 4’,6-diamidino-2-phenylindole was utilized for nuclear staining (Thermo Scientific).

qRT-PCR

To analyze organ-specific gene expressions, qRT-PCR was performed. Total RNA of the right cerebral hemisphere, lungs, liver, and kidneys were extracted using TRIzol reagent (Life Technologies, Carlsbad, CA, USA). cDNA was synthesized from the total RNA using the SuperScript III First-Strand Synthesis System (Invitrogen). Real-time PCR was performed using the FastStart Universal SYBR Green Master Mix (Roche) and Rotor-Gene Q (Qiagen, Venlo, Netherlands). The primer pairs are provided as supplemental data (Supplementary Table S2). Data analysis was performed using the 2−ΔΔCt method for relative quantification, with a Gapdh internal control.

BAL fluid analysis

To obtain BAL fluid from mice7, a plastic-coated 24-gauge needle was inserted into their trachea, and the needle was secured in place with elasticated thread. 1.0 mL of PBS was then instilled, and the lavages were retrieved. The lavages were subsequently centrifuged at 350 g for five minutes, and the supernatants were removed. The cell pellets were then combined in 0.2 mL of PBS and stained with Giemsa.

Statistical analysis

All data were analyzed using R software (version 4.03). Statistical significance was determined using Student’s t-test or analysis of variance with Tukey’s post hoc test for multiple comparisons, and the log-rank test for survival analysis. For all analyses, statistical significance was set at P < 0.05.

Supplementary Information

Supplementary Information.

Abbreviations

ALT Alanine transaminase

ANOVA Analysis of variance

AST Aspartate transaminase

BBB Blood-brain barrier

BUN Blood urea nitrogen

CD Cluster of differentiation

Cxcr2 C-X-C motif chemokine receptor 2

DAPI 4’,6-Diamidino-2-phenylindole

DAMPs Damage-associated molecular patterns

GFAP Glial fibrillary acidic protein

HMGB1 High-mobility group box 1

H&E Hematoxylin and eosin

Iba1 Ionized calcium-binding adapter molecule 1

IL Interleukin

KB Klüver-Barrera

Ly-6G Lymphocyte antigen 6G

MDSCs Myeloid-derived suppressor cells

NSS Neurological Severity Score

qRT-PCR Quantitative reverse transcription polymerase chain reaction

SEM Standard error of the mean

TBI Traumatic brain injury

TNF-α Tumor necrosis factor-α

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-72318-x.

We gratefully acknowledge Kanako Maruo and Kumiko Kokuryo for technical assistance. Furthermore, we thank Editage for their careful reading of the manuscript and for English editing. We also acknowledge the technical assistance and histopathological analyses performed by the members of the Center for Anatomical, Pathological, and Forensic Research at the Graduate School of Medicine, Kyoto University. This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI, grant number 21K17324 (CK).

Author contributions

Conceived and designed the experiments: C.K. and Y.N. Performed the experiments: C.K., M.M, and H.K. Analyzed the data: C.K., M.M., H.K., H.M., H.A., and K.T. Contributed reagents/materials/analysis tools: H.M. and H.A. Wrote the paper: C.K., M.M., and Y.N. All authors read and approved the final manuscript.

Data availability

The data that support the findings of the present study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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