Long wavelength light exposure reduces systemic inflammation coagulopathy, and acute organ injury following polytrauma in mice

Abstract

Background:

Evidence suggests that variation in light exposure strongly influences the dynamic of inflammation, coagulation, and the immune system. Polytrauma induces systemic inflammation that can lead to end-organ injury. Here, we hypothesize that alterations in light exposure influence post-trauma inflammation, coagulopathy, and end-organ injury.

Methods:

Study Type: Original Research Article. Level of Evidence: Basic Science (Level IV). C57BL/6 mice underwent a validated polytrauma and hemorrhage model performed following 72 hours of exposure to red (617nm, 1,700lux), blue (321nm, 1,700lux), and fluorescent white light (300lux) (n = 6–8/group). The animals were sacrificed at 6h post-trauma. Plasma samples were evaluated and compared for pro-inflammatory cytokine expression levels, coagulation parameters, markers of liver and renal injury, and histological changes (Carstairs staining). One-way ANOVA statistical tests were applied to compare study groups.

Results:

Pre-exposure to long-wavelength red light significantly reduced the inflammatory response at 6 hours post-polytrauma compared to blue and ambient light, as evidenced by decreased levels of IL-6, MCP-1 (both p < 0.001), liver injury markers (ALT, p < 0.05), and kidney injury markers (cystatin C, p < 0.01). Additionally, Carstairs staining of organ tissues revealed milder histological changes in the red light-exposed group, indicating reduced end-organ damage. Furthermore, PT was significantly lower (p < 0.001) and fibrinogen levels were better maintained (p < 0.01) in the red light-exposed mice compared to those exposed to blue and ambient light.

Conclusion:

Prophylactic light exposure can be optimized to reduce systemic inflammation, coagulopathy and minimize acute organ injury following polytrauma. Understanding the mechanisms by which light exposure attenuates inflammation may provide a novel strategy to reducing trauma related morbidity.

Background

Traumatic injury remains a prominent contributor to illness and death on a global scale (1). Severe trauma elicits a myriad of systemic responses. Specifically, trauma instigates the release of various substances, including tissue factor and cytokines that trigger both inflammatory and coagulative pathways (2). This intricate interplay between the immune and coagulation systems is foundational to comprehending the multifaceted responses post-trauma. For example, pro-inflammatory cytokines can be instrumental in enhancing thrombin generation, which in turn can activate further inflammatory processes. Additionally, platelets that undergo degranulation also enhance immune responses by activating neutrophils (3)(4). Furthermore, trauma-induced dysregulation in the immune response can influence hemostasis, exemplified by instances of fibrinolytic activation seen in certain trauma patients (5).

The consequences of trauma go beyond the initial injury, affecting both the immune and coagulation systems in a manner that requires a comprehensive understanding for optimal patient management. In the initial stages, hemorrhage is the primary factor responsible for early mortality. Nonetheless, those who manage to survive the initial trauma face the possibility of microvascular inflammatory complications, which can subsequently lead to organ damage (6, 7). In parallel, inflammation activation can potentiate the coagulation process through interactions with the innate immune system (8, 9). Traumatic injury results in innate immune system activation and inflammation resulting in a dysregulation of these systems and ultimately a thromboinflammatory response and trauma induced coagulopathy (7, 10). Identifying a way to augment the thromboinflammatory response to trauma without impairing hemostasis represents a novel therapeutic target that could improve outcomes following traumatic injury.

The anti-inflammatory effects of light exposure are gaining more attention, and the effect of optimizing light exposure following critical illness has been shown to improve outcomes (11). Modifying the spectrum of light exposure also impacts outcomes in other pro-inflammatory injuries such as ischemia/reperfusion (11). Red light therapy is becoming increasingly recognized for its efficacy in tissue preservation and stimulation of cellular function following different types of injuries. Currently, its main applications include promoting wound healing, reducing inflammation and edema, providing pain relief, and preventing tissue damagen(12, 13). In tandem with these advancements, light phototherapy (photobiomodulation) that utilizes long-wavelength light-emitting diodes (LEDs) is employed in the treatment of a broad spectrum of medical conditions. It is particularly effective in managing pro-inflammatory responses such as enhancing wound healing and assisting post-surgical recovery (14, 15). Importantly, long-wavelength red light has demonstrated anti-inflammatory capabilities in a model of ischemic colitis (16), further underlining its potential for immunomodulation of the inflammatory response. In addition to its inflammatory modulation, studies have explored the effects of red light on coagulation. Notably, one study demonstrated that far-red light independently inhibits platelet activity and clotting (17).

The potential therapeutic benefits of modifying light exposure in mitigating trauma-induced complications has garnered increasing attention (5). To date, no studies have examined the impact of light exposure on outcomes following injury, particularly the unique influences of post-trauma thromboinflammation on organ damage. We hypothesized that enhanced exposure to high-wavelength red light prior to injury would attenuate inflammation.

Materials and Methods

Light exposure

All experiments took place in a room maintaining a 12-hour light and 12-hour dark cycle, with a constant temperature of approximately 25 ± 2°C. Three types of light (red, blue, and ambient white) were tested. The light sources, equipped with blue and red filters, ensured consistent illumination of approximately 1400 lux at the cage centers, confirmed through previous research (18) . Additionally, a control group experienced ambient white, fluorescent lighting at 400 lux without a dominant wavelength spectrum. Intensity of light was measured with lumens from a handheld digital lux meter (Digital Light Level Meter LX1330B, Mastech, Dongguan, Guangdong, China). The lighting did not significantly affect room or cage temperatures as previously described.(19) Mice were exposed to one of the light conditions for 72 hours prior to polytrauma and received an additional 6 hours of light exposure during the recovery phase before being sacrificed (Figure 1).

Figure 1.

The Spectrum of Light Exposure and Study Design: This figure provides a schematic representation of the study, detailing the methods used for subjecting the animals to the selected lighting conditions and the intricacies of the animal modeling.

Animal Housing

C57BL/6J mice were sourced from Jackson Laboratories (Bar Harbor, ME). The mice were housed in compliance with local Animal Research and Care Committee and with the animal welfare guidelines at the National Institutes of Health (Bethesda, MD, USA). Our study complied with the ARRIVE guidelines (20). The guideline governs the reporting of in vivo animal experiments, ensuring that our methods and results are both transparent and thorough in their presentation.

Experimental design

A total of 32 male C57B/l6 mice, aged 10 weeks and weighing between 25–31 g, were used. Each experimental group consisted of 6 animals. The sample size for each group (6 animals) in this study was selected based on common practices in similar investigations focused on the effects of light exposure and polytrauma on physiological parameters ensuring sufficient statistical power to detect significant differences (18, 21). Mice were exposed to varied light for 72 hours and then subjected to either polytrauma or sham experiments. Following surgical intervention, mice were exposed to an additional 6 hours of light during recovery before sample collection and analysis.

Mouse Model of Polytrauma and Hemorrhagic Shock

Anesthesia was induced using 5% isoflurane in room air via a nosecone and maintained with 2% to 2.5% isoflurane until the end of polytrauma or sham procedures. For analgesia, buprenorphine was injected subcutaneously at 0.05 mg/kg bodyweight prior to polytrauma induction. Polytrauma and hemorrhagic shock was induced in mice using a comprehensive approach over a span of 15 minutes as previously described (8, 21). First, to simulate hemorrhagic shock, acute blood loss was induced through blind cardiac puncture using a 1 mL syringe with a 30G needle, with approximately 25% of the total blood volume drawn. Subsequently, polytrauma was induced using a combination of pseudofracture and soft liver injury. The pseudofracture model, which consists of both soft tissue injury and injection of crushed bone solution, was achieved by placing a hemostat around the posterior thigh musculature, followed by the injection of 0.15 mL of crushed bone solution obtained from an age, weight, and sex-matched donor as previously described(22) (Figure 1). To further intensify the severity of the polytrauma model, a laparotomy procedure was performed. Specifically, the left middle lobe of the liver was targeted using sterile forceps and was crushed three times using a sterile hemostat to ensure a consistent and reproducible injury across experiments. This method induces a significant but controlled injury to the liver lobe. These combined steps aimed to emulate the multifaceted nature of polytrauma and facilitate investigations into the pathophysiological responses and potential therapeutic interventions associated with polytrauma and hemorrhagic shock in mice (Figure 1). After the procedure, animals were returned to their cages and placed under the same designated lighting conditions as before the start of the procedure. No resuscitation was performed post-procedure. Animals were closely monitored for behavioral changes throughout the experiment and were euthanized 6 hours later. For sham experiments, animals underwent anesthesia and a laparotomy procedure, similar to the experimental group, to eliminate potential confounding effects of anesthesia and laparotomy on inflammatory markers and other study parameters. Following the laparotomy, their incisions were closed, and they were returned to their cages. These animals were then sacrificed 6 hours later for further analysis.

Animals were monitored for signs of severe distress or complications, such as uncontrolled bleeding, due to the polytrauma or sham models. The observed mortality rate of 10%, occurred exclusively as a result of the procedures. Once animals survived the immediate procedure, none met the exclusion criteria. Consequently, data from all these surviving animals were included in the analysis. This underscores that our approach was relatively safe and well-tolerated for the animals post-procedure. In our study, trained personnel ensured standardized modeling procedures. We employed blinding techniques to reduce biases in data collection and analysis, maintaining consistent sample collections and post-procedure measurements after 6 hours. Additionally, efforts were made throughout to limit awareness of group allocation, preserving the integrity of our research..

Tail Bleeding Assays

After 72 hours of light exposure, uninjured mice, previously exposed to either red or ambient light, underwent tail tip transection to quantify tail bleeding times as we have previously described (23). For this experiment, a total of 20 mice were used, divided into two groups of 10 animals each. The mice were anesthetized using Nembutal, followed by a 10 mm incision at the tail’s end using a scalpel. The tail was then submerged in a 50 ml conical tube filled with saline warmed to 37 degrees Celsius. The bleeding time was recorded as the duration until the bleeding ceased (24).

Tissue Staining for Microthrombosis Visualization

Tissues from the liver and kidney, obtained through perfusion fixation and cryopreservation, were sectioned into 5-μm thick serial slices. Emphasizing the visualization of microthrombosis, these sections were processed using Carstairs’ staining (25). The staining procedure comprised mordanting in ferric ammonium sulfate, staining with Mayer’s hematoxylin, exposure to picric acid-orange G, subsequent staining with ponceau fuchsin, differentiation using phosphotungstic acid, and a final treatment with aniline blue prior to mounting. Fibrin is presented as bright red and red blood cells appeared yellow.

Measurements of cytokine concentrations and circulating Myeloperoxidase (MPO)-DNA complexes

IL-6 and MCP-1 concentrations in plasma were measured by using commercially available mouse IL-6 and MCP-1 ELISA kits (ThermoFisher Scientific, Waltham, MA, USA). The levels of myeloperoxidase (MPO)-DNA complexes in the plasma of mice were quantified using an enzyme-linked immunosorbent assay (ELISA) kit (ThermoFisher Scientific, Waltham, MA, USA). All assays were performed in duplicate according to the manufacturer’s instructions. Cytokine levels were determined by comparing the optical density results to standard curves using recombinant cytokines, provided by the manufacturer.

Measurement of plasma coagulation factor activity

The STA-NEOPLASTINa and STA-PTT AUTOMATE kit (Diagnostica Stago, Asnieres, France) was employed to measure prothrombin times (PT) and activated partial thromboplastin time (aPTT), respectively. Plasma fibrinogen levels were determined using the Fibri-Prest Automate Kit (Diagnostica Stago). All assays were conducted using the Start-8 Coagulation Analyzer (Diagnostica Stago). To assess the levels of thrombin and thrombin-antithrombin complex (TAT) in plasma, a TAT enzyme-linked immunosorbent assay (ELISA) kit (Abcam, Cambridge, UK) was employed.

Assessment of End-Organ Damage:

Liver injury was assessed by measuring the levels of serum alanine transaminase (ALT) and aspartate transaminase (AST) using a Heska DC7000 Dri-chem analyzer (Heska Corporation, Loveland, CO, USA). The concentration of Cystatin C as a marker of acute kidney injury in the serum was determined using an enzyme-linked immunosorbent assay (ELISA) kit (ThermoFisher Scientific, Waltham, MA, USA)(21). Cystatin C was selected as the primary marker for acute kidney injury (AKI) due to its superior sensitivity and specificity in early AKI detection compared to serum creatinine. Studies have underscored Cystatin C’s utility, especially when assessing AKI within short post-trauma windows (26, 27). This is particularly relevant for the current study’s methodology, as animals were sacrificed just 6 hours post-trauma, a timeframe where traditional markers such as serum creatinine may not yet accurately reflect glomerular filtration rate changes (28).

Statistical Analysis

Statistical analysis was conducted using Prism 9.0 software (GraphPad Software, San Diego, US). ANOVA was utilized for most measurements, with the t-test for bleeding time. Based on preliminary data and prior experiments with similar models, a power analysis was performed. It determined a sample size of 6 animals per group would provide over 80% power to detect significant differences, using an alpha of 0.05. Data normality was confirmed with the Shapiro-Wilk test, justifying representation as mean ± standard deviation. Statistical significance was set at P < 0.05, and one-way ANOVA with Tukey’s multiple comparison test was employed.

Results:

Behavioral Observations

Throughout the study, no noticeable behavioral changes were observed in animals across all light exposure groups, indicating that the light exposures did not induce any discernible stress or behavioral alterations in the subjects.

Blood Coagulation

Blood samples were collected at 6 hours post-induction of polytrauma and hemorrhagic models. PT was significantly lower in red light-exposed mice than in blue light-exposed mice (PT: Red 13.97 ± 3.27 seconds, Blue 52.95 ± 19.34 seconds; P = 0.0006) after injury.

There were no significant differences between red light-exposed mice compared to the sham group, while both the blue and ambient groups showed prolonged PT times when compared to the sham group (PT: Blue 52.95 ± 19.34 seconds, Ambient 36.27 ± 19.99 seconds, Sham 10.03 ± 1.42 seconds; P = 0.0002 and P = 0.0197, respectively) (Figure 2A).

Figure 2:

Effects of Light Exposure on Coagulation Parameters and Bleeding Times: At 6 hours post-polytrauma, blood samples were collected from six subjects under anesthesia via cardiac puncture. Analyses were conducted to determine PT (A), Fibrinogen (B), and aPTT (C) levels. Post 72 hours of light exposure, tail-tip transections were performed on ten subjects, and the bleeding times (D) were recorded. This time measures from the onset of a 10mm incision to bleeding cessation, including any rebleeding events within 30 seconds. The presented data reflects means ± SD. PT, Prothrombin Time; aPTT, Activated Partial Thromboplastin Time; SD, Standard Deviation. The number of stars accompanying p-values in the legend indicates the level of significance: (*) signifies p < 0.05, (**) p < 0.01, (***) p < 0.001, and (****) p < 0.0001, representing the chance of results occurring by random chance.

At 6 hours post-polytrauma, fibrinogen levels markedly decreased in blue and ambient light groups, while, strikingly, red light-exposed animals maintained their fibrinogen levels with a statistically smaller decrease relative to sham (Figure 2B) (Fibrinogen: Red 1.42 ± 0.22 g/l, Blue 0.56 ± 0.34 g/l, Ambient 0.60 ± 0.23 g/l; P = 0.0001 and P = 0.0003, respectively).

To test the impact of light exposure specifically on hemostasis, a murine tail bleed assay was performed as previously described (24). Bleeding times were not significantly different between the red light and ambient light groups after 72 hours of exposure suggesting no discernible impact of light color on the rate of hemostasis (Figure 2D). In addition, there was no significant difference in the duration of aPTT six hours post-polytrauma between the groups exposed to red ambient light and blue light (Figure 2C).

Influence on inflammatory markers IL-6, MCP-1 and MPO

Pre-exposure to red light attenuated trauma-induced IL-6 elevation at 6 hours post-polytrauma compared to that of the blue light (IL-6: Red: 89.17 ± 36 pg/ml, Blue: 231.23 ± 134 pg/ml; P = 0.0426). Additionally, the plasma level of IL-6 in red-light exposed mice was not significantly different from that of the sham group (Figure 3A).

Figure 3.

Impact of Light Exposure on Inflammatory Markers 6 Hours Post-Polytrauma and Hemorrhagic Shock: Blood samples were taken from six subjects under anesthesia via cardiac puncture at 6 hours post-polytrauma. The provided values represent means ± SD for IL-6 (A), MCP-1 (B), and MPO (C). IL-6, Interleukin-6; MCP-1, Monocyte Chemoattractant Protein-1; MPO, Myeloperoxidase; SD, Standard Deviation. The number of stars accompanying p-values in the legend indicates the level of significance: (*) signifies p < 0.05, (**) p < 0.01, (***) p < 0.001, and (****) p < 0.0001, representing the chance of results occurring by random chance.

Red light exposure resulted in lower plasma concentrations of MCP-1 than the blue and ambient groups (MCP-1: Red: 214.33 ± 48.86 pg/ml, Blue: 350.03 ± 28 pg/ml, Ambient: 408.98 ± 78.35 pg/ml; P = 0.0007 and P < 0.0001, respectively). However, all the groups exhibited significantly higher concentrations when compared to the sham group (Figure 3B), indicating the inflammation induced by the model.

Myeloperoxidase (MPO) is an enzyme present in neutrophils that plays a vital role in combating pathogens, and its elevated levels are often associated with trauma. (29). The MPO protein levels were significantly lower in the Red-light group compared to the ambient and blue groups (MPO: Red: 13.22 ± 4.9 ng/ml, Ambient: 22.92 ± 3.49 ng/ml, Blue: 20.03 ± 5.57 ng/ml; P = 0.0036 and P =0.0493, respectively) Importantly, no significant differences were found between the sham group and the red-light group (Figure 3C).

Effect of Light on Organ Injury and Histological Evidence of Tissue Damage

Mice exposed to red light showed significantly reduced serum levels of AST and ALT six hours after polytrauma compared to the ambient light group (AST: Red: 1688.3 ± 545 IU/ml, Ambient: 2198.33 ± 246 IU/ml; P = 0.0487; ALT: Red: 3105.00 ± 1193 IU/ml, Ambient: 4851.67 ± 1026 IU/ml; P = 0.0082) (Figure 4A and 4B). Additionally, the red light-exposed group exhibited lower levels of Cystatin C compared to the ambient group, indicating reduced end organ damage (Cystatin C: Red: 2758.73 ± 250 IU/ml, Ambient: 3473.60 ± 328 IU/ml; P = 0.0081). Notably, all polytrauma groups showed higher levels of Cystatin C compared to the sham group (Figure 4C). To further understand the observed differences in blood coagulation factors and serum markers of organ injury, we examined the kidney and liver tissues under Carstairs stain, which is particularly effective at highlighting fibrin deposition and microthrombosis. Figure 4D presents the stained sections of kidney and liver tissues harvested 6 hours post-polytrauma from both red light pre-treated and ambient-light exposed animals. Observations in the kidney (displayed in the top row) revealed that ambient-light exposure led to pronounced fibrin deposition in the glomeruli (as indicated by dark short arrows) and evident microthrombosis (marked by golden asterisks). Similarly, in the liver sections (shown in the bottom row), microthrombosis was prominently observed in the sinusoids, as marked by golden asterisks.

Figure 4.

Effects of Light Exposure on Liver and Kidney Function and Histological Changes 6 Hours After Polytrauma and Hemorrhagic Shock: Blood samples were collected from six subjects under anesthesia via cardiac puncture 6 hours post-polytrauma. Displayed are the means ± SD values for AST (A), ALT (B), and Cystatin C (C). Additionally, sections from red light pre-treated and ambient-light exposed animals show histological changes. The kidney section (D, top row) reveals fibrin deposition in the glomeruli (indicated by a dark short arrow) and the presence of microthrombosis by a golden asterisk (*). In the liver section (D, bottom row), microthrombosis in the sinusoids is denoted by a golden asterisk (*). AST, Aspartate Aminotransferase; ALT, Alanine Aminotransferase; SD, Standard Deviation. The number of stars accompanying p-values in the legend indicates the level of significance: (*) signifies p < 0.05, (**) p < 0.01, (***) p < 0.001, and (****) p < 0.0001, representing the chance of results occurring by random chance.

Discussion

In the early stages of trauma, inflammation and coagulation disruptions are interconnected and ultimately contribute to end-organ failure. Exposure to various wavelengths of light is associated with anti-inflammatory and coagulation-modulating effects. In the present study, using a murine trauma model, we demonstrate that subjecting the animals to 72 hours of pre-exposure to long-wavelength red light, combined with continued exposure during recovery, yields a powerful anti-inflammatory response. The wavelength of light also impacts coagulation regulation following injury, and likely a combination of the anti-inflammatory effect and positive impact on trauma induced coagulopathy leads to diminished end-organ injury observed 6 hours after the trauma.

The present study, which explores the effects of blue, red, and ambient light exposure on small animal polytrauma, demonstrates that red light exposure significantly attenuates the trauma-induced pro-inflammatory response. This is achieved by reducing the systemic expression of the key pro-inflammatory cytokines IL-6 and MCP-1 (30). Long wavelength red light has been shown to reduce inflammation by decreasing neutrophil influx, oxidative stress, and edema in a dose-dependent manner (12)(13)(14). Studies have also demonstrated the regional effect of red light in controlling local inflammation in conditions such as wound healing (31) and inflammatory bowel disease (16) and inflammatory pain (12). In line with this, our study shows that pre-exposure to long wavelengths, and not blue nor ambient light, can result in a reduction in MPO levels in the polytrauma mice. This reduction in MPO levels supports our observation of reduced IL-6 and MCP-1 levels, indicating a potential modulation of the immune response and inflammation caused by trauma. While the benefits of systemic redlight therapy on inflammation have been reported, the present analysis is novel in the application of red light to thromboinflammation after severe systemic inflammation due to trauma.

Our study demonstrated that the application of red light effectively mitigated trauma-induced coagulopathy as measured by reducing the PT, aPTT, and fibrinogen consumption. Concurrently, histological examinations, aligned with data indicating end-organ damage, showed a distinct advantage in the red light pre-treated group. The combined evidence accentuates the multifaceted protective impact of red light therapy, especially in guarding against trauma-induced organ injuries. There are limited studies investigating the effects of red light on blood coagulation characteristics. In one study, the combination of far-red light and nitrite treatment consistently reduced coagulation measures, including platelet adhesion inhibition, whereas individual treatments far-red light alone showed mixed results and, in some cases, had no effect (17). Other studies have demonstrated that exposing blood to long wavelength red light in vitro led to a time-dependent reduction in platelet adhesion and aggregation under shear stress conditions (32) (33). Although we have not directly evaluated platelet function in our current study, this is the subject of active investigation and is an important area of future research.

Long-wavelength red light has many potential mechanistic effects in reducing inflammation and coagulation post-trauma. At the cellular level, long wavelength light has been shown to reduce platelet activation and consumption ex vivo (34). On a molecular level, red light has been linked with photobiomodulation, which affects signaling pathways such as the mitochondrial respiratory chain. This modulation can lead to enhanced ATP production, reduced oxidative stress, and adjustments in intracellular calcium levels, all integral to inflammation control(35). Furthermore, the role of red light in influencing circadian rhythms offers an additional mechanism of action. Disruptions in circadian patterns have been known to exacerbate inflammatory responses, suggesting that red light’s ability to regulate these rhythms might contribute to its observed anti-inflammatory effects(36).Together, these cellular to molecular mechanisms emphasize the diverse impacts of red light in potentially alleviating inflammation.

This study provides valuable preliminary data; however, it also presents several significant limitations. The use of murine model, while a practical and a widely used choice in early research stages, may not be directly applicable to human physiology. Larger animal models and, in due course, human trials would provide a more definitive understanding of the relevance of our findings. In the present study, we used only male mice. We are aware of the critical importance of sex as a biologic variable. However, the impact of sex upon outcomes in murine models of trauma and the unknown impact of sex on light effects would have necessitated a substantial increase in the number of animals for these exploratory studies. Further, female specific hormones such as estrogen may influence coagulation and hemostasis so limiting this variable in the current study addressed this potential confounder (37). However, we acknowledge the importance of including both sexes whenever possible for a comprehensive understanding of our research. The precise mechanism of action underpinning the impact of red light remains undefined, though it is currently under rigorous investigation. As this knowledge gap is bridged, it is anticipated that this will not only shed light on the exact mechanisms at play but also inform the development of future approaches to managing trauma-induced inflammation. Our study also focused on the effect of long-duration pre-exposure to red light. This decision was based on preliminary data from mechanistic studies, with the recognition that shorter durations might not have the same effects (19). Despite rigorous measures to curtail biases, such as blinding and allocation, inherent subjectivity persists in our study’s nature and modeling. Additionally, our experimental design, which implemented a fixed volume hemorrhage and was conducted on uninjured animals, limited our ability to specifically assess the impact of red light on hemostasis. This choice of using uninjured animals poses a limitation in the direct applicability of the findings. To address some of these concerns, we have incorporated a tail vein bleed assay, a well-characterized murine hemostasis test, which demonstrates that the spectrum of light does not negatively affect hemostasis in this context. Moving forward, it is essential for future studies to offer a more comprehensive examination of the mechanism by which light augments post-traumatic thromboinflammation. This would provide a deeper insight into the potential preventive role of red light in trauma-induced coagulopathy. Furthermore, it is crucial to emphasize that further investigations should explore the relative benefits of pre-injury treatment versus post-injury treatment, and whether post-injury treatment alone could have benefits or even augment the anti-inflammatory effects of pre-injury treatment, particularly considering the unique influences of post-trauma thromboinflammation on organ injury.”

In conclusion, the present study provides intriguing observations regarding the potential impact of red-light pre-exposure on post-traumatic inflammation, coagulation, and end-organ damage in a murine trauma model. The observed reductions in MCP-1, IL-6, and MPO levels, along with improved coagulation parameters align with our histological findings, further confirming the protective benefits of red-light therapy against organ injuries. While the results are promising, our findings serve as an initial observation that is hypothesis generating, and additional research is imperative to establish a causal relationship and understand the mechanisms by which altering the wavelength of light exposure mitigates thromboinflammation.