Introduction

Hemorrhagic shock (HS) can cause death due to hypoperfusion and progressive inflammation, which may lead to multiple organ dysfunction syndrome (MODS) (1). Several studies have shown the effects of liver and kidney dysfunction post-hemorrhagic shock (2,3). The dysfunction mechanism in these two organs begins with tissue injury; however, the progression of injury between the two differs. Liver injury progression occurs due to circulating pro-inflammatory factors that concentrate at the injury site (4), while kidney injury progression results from the vulnerability of renal tubules to hypoxia, exacerbated by intra-renal vasoconstriction caused by sympathetic activity stimulation and renin-angiotensin system activation (5). The key to protecting organs from damage post-hemorrhagic shock is suppressing the activation and circulation of inflammatory mediators. Hemorrhagic shock requires prompt and effective intervention. Fluid therapy is a primary approach to restore intravascular volume and maintain organ perfusion in critically injured patients (6). The limitations of fluid therapy alone in managing hemorrhagic shock have been demonstrated in previous studies. Although fluid resuscitation restores systemic hemodynamics, it fails to prevent reperfusion-related cellular damage and may even contribute to endothelial injury and organ dysfunction (5,7). Inappropriate fluid volume or composition may trigger inflammatory cascades and worsen vascular leakage (8). To address these limitations, researchers have investigated pharmacological adjuncts such as dexamethasone. Administration of dexamethasone during hemorrhagic or traumatic injury was shown to reduce leukocyte infiltration, support vascular integrity, and improve clinical outcomes (9,10). Dexamethasone also contributed to organ protection by modulating inflammation and preserving endothelial function (11). However, potential drawbacks, such as hyperglycemia and altered metabolism at higher doses (12). These findings suggest that while fluid therapy and dexamethasone offer benefits, their combined limitations justify the exploration of alternative strategies, such as non-invasive neuromodulation approaches. The vagus nerve, one of the cranial nerves, plays a critical role in regulating inflammation. Studies have shown that vagus nerve stimulation (VNS) activates the anti-inflammatory cholinergic pathway (13). VNS studies related to HS indicate that vagus nerve stimulation during HS can enhance hemostasis, improve blood flow, and reduce inflammatory responses (14-16). This study employs a different VNS approach compared to previous research, which utilized invasive electrical stimulation targeting the nerve (7-9). In this research, ultrasound therapy (UST) is used for stimulation. UST was chosen because several studies have demonstrated its ability to influence vagus nerve activity (neuromodulation) (17). UST operates through three mechanisms: thermal, cavitation, and mechanical. These mechanisms induce changes in neural action potentials (18).

Ultrasound therapy offers advantages such as ease of use and non-invasiveness, making it a promising first-aid treatment for hemorrhage that does not require specialized expertise. To date, no studies have explored the benefits of ultrasound therapy as a vagus nerve stimulator for treating HS and preventing multiorgan dysfunction. In this study, UST is employed as a supporting therapy to prevent organ damage by suppressing inflammatory activity post-hemorrhagic shock.

Materials and methods

Ethical approval

All procedures conducted in this study were approved by the School of Veterinary Medicine and Biomedical Sciences (SVMBS) IPB University Animal Ethics Committee with the number 033/KEH/SKE/III/2023.

Research methodology

This study used 21 male New Zealand White rabbits weighing 2–3 kg, divided into seven groups: sham, UST, UST+Dx, FT+UST+Dx, FT+UST, FT+Dx, and FT, respectively. All groups were subjected to inhalation anesthesia with isoflurane. All Groups were induced with hemorrhagic shock (HS) by withdrawing 30–35% of the total blood volume (60 ml x body weight) through the auricular artery over 15 minutes. The induction method used in this study was a fixed-volume hemorrhagic shock (19).

The ultrasound device used in this study was the Hanil HS-502 (Hanil-TM Co., Ltd., Korea), a physiotherapeutic unit operating at 1 MHz, equipped with a crystal vibrator probe (effective radiating area ~4 cm²), and capable of continuous or pulsed output at adjustable intensities from 0.1 to 1.5 W/cm². Ultrasound therapy targeting the vagus nerve was applied to the ventral cervical region every 30 minutes for 10 minutes at a frequency of 1 MHz and an intensity of 1 W/cm². These parameters were selected based on ultrasound therapeutic for deep tissue effects (20), and supported by previous studies (21-23) demonstrated beneficial anti-inflammatory, cardioprotective, and immunomodulatory effects using ultrasound with comparable settings. The fluid therapy used in this study refers to hypotensive resuscitation with lactated Ringer’s solution, combined with intramuscular administration of dexamethasone (5 mg/mL) at a dose of 0.6 mg/kg.

Blood chemistry was evaluated by comparing baseline values, shock at minute 60, and final values at minute 180. The tests included alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate aminotransferase (AST), total protein (TP), blood urea nitrogen (BUN), and creatinine (Cr), measured using a blood chemical analyzer (Arkray® SPOTCHEM EZ SP-4430). After 180 minutes, euthanasia was performed using an overdose of anesthesia, followed by the collection of liver and kidney tissues for histopathological examination using hematoxylin and eosin (H&E) staining.

Data analysis

To determine changes in blood chemistry post-hemorrhagic shock, blood chemistry values at minute 0 were compared to those at minute 60. To evaluated differences in blood chemistry between treatment groups, blood chemistry values at minute 60 were compared to those at minute 180 in all groups (1–8). The data were analyzed using one-way ANOVA with Duncan's post hoc test. The blood chemistry variable values that had been statistically tested were then observed for patterns (decrease, increase < 60%, or > 60% from the value during hemorrhagic shock). Variables that showed a decrease were given a score of 3, an increase < 60% was given a score of 2, and > 60% was given a score of 1. The scores for each group per variable were then summed and depicted in a graph.

Histopathological examinations were performed to evaluated tissue damage in both liver and kidney using a semiquantitative scoring method adapted from previous studies (24-26). In the liver, three variables were evaluated: hepatocyte degeneration, vascular and sinusoidal congestion, and hepatocyte apoptosis. Observations were conducted in zone 3 (central vein area), with degeneration evaluated at 20× magnification and congestion and apoptosis at 40× magnification. The area of degeneration was measured, while hepatocyte counts for congestion and apoptosis were quantified using ImageJ® software. In the kidney, histopathological examination focused on the cortical area, where four pathological changes were evaluated: tubular epithelial edema, vascular congestion, Bowman’s space dilatation, and hyaline leakage. All variables were observed at 20× magnification, and quantitative analysis was performed using ImageJ® software. Lesions and normal areas in the proximal tubules and glomeruli were calculated to determine percentages. Each variable was then scored based on the percentage of affected cells: 0 for 0%, 1 for 1–25%, 2 for 26–50%, and 3 for >50%. Histopathological scoring data were statistically analyzed using the Kruskal-Wallis and Mann-Whitney tests. Final scores from liver and kidney examinations were ranked to evaluate protective effects.

Results

Blood chemistry in shock condition

Induction of blood loss in Groups 1-7 resulted in significant changes (p < 0.05) in the blood chemistry parameters total protein (TP), blood urea nitrogen (BUN), and creatinine (Cr), while changes in alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were not statistically significant (p > 0.05) (Figure 1). Compared to baseline (minute 0), levels of ALP, AST, BUN, and Cr increased, whereas ALT and TP levels decreased. Furthermore, when compared to the normal reference values for average rabbits the levels of ALP, ALT, AST, and Cr in the shock condition were above the normal range, while TP and BUN were below the normal range.

Figure 1: Blood chemistry in hemorrhagic shock

Blood chemistry after treatment

Treatment affected blood chemistry values (Figure 2). Groups FT+UST+Dx, FT+Dx, and FT showed a decrease in ALP and ALT values. No treatment group showed a decrease in AST values (Figure 2). However, FT+UST+Dx, FT+Dx, and FT caused a slight increase (< 60%), while the others caused a significant increase (> 60%) in AST (Figure 3A). The protective effect on the liver in this study was evaluated based on blood chemistry values of ALP, ALT, and AST. Ranking the treatment groups from those with positive to negative effects (decrease, slight increase, and significant increase), the order of liver protection based on blood chemistry values is FT+UST+Dx, FT, FT+Dx, FT+UST, UST+Dx, and UST (Figure 3B).

Treatment groups involving FT without UST showed a decrease in TP and Cr values, while FT combined with UST showed a decrease in TP and an increase in Cr. All treatment groups showed an increase in BUN values. Groups with FT combinations showed a slight increase in BUN values compared to groups without FT combinations. Groups without FT (UST+Dx and UST) showed an increase in TP, BUN, and Cr values (Figure 2). The protective effect on kidney damage in this study was evaluated based on blood chemistry values of TP, BUN, and Cr. Ranking the treatment groups from those with positive to negative effects (decrease, slight increase, and significant increase), the order of kidney protection based on blood chemistry values is FT+Dx and FT, FT+UST+Dx and FT+UST, UST+Dx, and UST (Figure 4).

Figure 2: Blood chemistry after treatment.

Figure 3: The effect of therapy on liver blood chemistry variables. 3A. Plot liver blood chemistry values as function of therapy. Circle: decrease effect (score 3), square: slight increase (< 60%) (score 2), triangle: significant increase (> 60%) (score 1). 3B. Rank liver blood chemistry scores as an effect of therapy.

Figure 4: The effect of therapy on kidney blood chemistry variables. 4A. Plot kidney blood chemistry values as an effect of therapy. Circle: decrease effect (score 3), square: slight increase (< 60%) (score 2), triangle: significant increase (> 60%) (score 1). 4B. Rank kidney blood chemistry scores as an effect of therapy

Histopathology

Based on the results of liver histopathological examination (Figure 5), hepatocyte degeneration was observed with scores ranging from 1.22 to 1.78 (1% < average < 25%), with the highest score in the FT group. Vascular and sinusoidal congestion were also found in all treatment groups with scores ranging from 1 to 2.22 (25% < average < 50%), with the highest score in the FT+Dx treatment. In contrast, apoptosis was in the range of scores from 0.14 to 1 (average < 25%), with the highest scores in the FT+UST+Dx and NTR groups. All treatment groups showed significant differences from the FT group (0.14 ± 0.24) (Table 1).

Figure 5: Histopathological features of liver tissue at the central vein margin by group. Apoptotic changes and sinusoidal congestion, scale bar 100 μm. 5A: Sham; 5B: UST group; 5C: UST+Dx group; 5D: FT+UST+Dx group; 5E: FT+UST group; 5F: FT+Dx group; 5G: FT group. Red arrows: degeneration; blue arrows: apoptosis; green arrows: congestion.

Table 1: Liver histopathological score

Note: Data are presented as mean with standard deviation (x ± SD). The same superscript letters in the same column indicate no significant difference (p > 0.05)

In the liver, hepatocyte degeneration and apoptosis were observed in up to 25% of the tissue, with congestion reaching up to 50%. The characteristic histopathological change in the liver following hemorrhage is generally bleeding; however, since the method used in this study involved fixed-volume blood withdrawal, the resulting liver changes were predominantly degenerative. Degenerative changes are commonly observed in hepatocytes due to increased Reactive Oxygen Species (ROS) production. The protective effect on the liver in this study was evaluated based on the number of pathological changes found in the organ of each treatment group, as reflected in the scoring system. When ranked from the group with the least damage to the most, the therapeutic protective capacity for the liver was as follows: FT+UST, FT, UST+Dx, FT+UST+Dx, FT+Dx, and UST (Figure 6).

Changes in the kidneys were observed based on four parameters, but only two parameters showed differences among several groups (Figure 7). In the kidney tissue, tubular swelling, Bowman’s space expansion, and hyaline leakage were observed in up to 25% of cases, and vascular congestion in up to 50%. The Bowman’s space expansion parameter, with scores ranging from 0.22 to 1.33, and hyaline leakage, with scores ranging from 0.00 to 0.33, showed no significant differences among groups (Table 2). Bowman’s space expansion had the highest score in the NTR group, while the lowest was found in the group with the Dx combination. The group receiving UST exhibited Bowman’s space expansion within the moderate to severe category. This finding is consistent with the effects of analgesic drugs that act by inhibiting neurotransmitter release, including glutamate, which have also been reported to induce similar renal changes. This suggests that UST may potentially cause nephrotoxic effects resembling the pattern of damage associated with certain analgesics.

Hyaline leakage had the highest score in the UST group. For tubular swelling, the NTR group showed the highest score of 1.67 with a range of scores from 1.00 to 1.67 (1% < average < 25%), and a significant difference from the FT+UST group. Based on the vascular congestion parameter, the score range was 0.00 to 2.22 (0 < average < 50%). The FT group had a score of zero, indicating no congestion observed, whereas the UST treatment group had the highest congestion score of 2.22 (Table 2). Significant differences were found between the FT group and the UST and NTR groups. When ranked from the group with the least damage to the most, the therapeutic protective abilities on the kidney organ are ranked as follows: FT+UST+Dx, UST+Dx, FT+UST, FT+Dx, FT, and UST (Figure 6).

Figure 6: Rank of protection score. 6A: Ranking of liver protection scores. 6B: Ranking of kidney protection scores.

Figure 7: Kidney histopathological appearance in the cortex by group. Tubular swelling and Bowman’s space dilation observed, bar 100 μm. 7ANTR group; 7B UST group; 7C UST+Dx group; 7D FT+UST+Dx group; 7E FT+UST group; 7F FT+Dx group; 7G FT group. Red arrow: Bowman’s space dilation, blue arrow: tubular swelling, green arrow: congestion, purple arrow: hyaline deposition.

Table 2: Kidney histopathological score

Note: Data are presented as mean with standard deviation (x ± SD). The same superscript letters in the same column indicate no significant difference (p > 0.05)

Discussion

Liver injury parameters in hemorrhagic shock

One of the effects of hemorrhagic shock (HS) on the liver is ischemia (hepatic ischemia). This occurs due to reduced vascular resistance (27-29). Ischemic hepatocytes subsequently lead to hepatocyte injury, characterized by mitochondrial dysfunction, excessive reactive oxygen species, and increased apoptosis (29). Hepatocyte injury, whether reversible or irreversible, induces the release of enzymes such as ALP, AST, ALT, and others like GLDH, SDH, LDH, and alpha-glutathione S-transferase (α-GST). The presence of these enzymes in the bloodstream does not always indicate cell death (30) and may also occur in conditions of increased oxidative stress and imbalance with the body’s antioxidant defenses (31). In this study, different results were observed. During shock conditions, ALP and AST levels increased, whereas ALT levels decreased. The increase in ALP and AST levels is attributed to HS-induced hypoxia, which leads to cell damage. Elevated ALP and AST levels indicate injury to cells, primarily in the liver. These values are typically observed in the acute phase of hepatocellular injury (30). ALP levels increased earlier because the release of this enzyme begins after hypoxia triggers anaerobic metabolic shifts, ATP depletion, membrane integrity disruption, and the release of pro-inflammatory agents, particularly IL-1, IL-6, and TNF-α (32). AST levels were observed to rise earlier than ALT levels, likely due to AST's broader distribution in organs such as skeletal and cardiac muscles, kidneys, and red blood cells (33). The decrease in ALT levels is likely due to its localization in the cytosol, with an isoenzyme mitochondrial ALT also found in mitochondria, particularly in the periportal zone of the liver (34). This zone is closest to the oxygen supply, resulting in a slower response to hypoxia (34).

Kidney parameters in shock conditions

Elevated BUN and creatinine (Cr) levels are key indicators of acute kidney injury (AKI) (35). The kidneys are among the most sensitive organs to hypoxic conditions, making them highly susceptible to tubular and endothelial hypoxia injuries that can lead to AKI (36). During hemorrhagic shock (HS), excessive blood loss disrupts both macro and microcirculation. These disruptions occur due to the slowed movement of red blood cells (RBCs) in the vasculature and a decrease in oncotic pressure, characterized by reduced vascular turgor (5).

During shock, systemic and renal autoregulatory mechanisms are activated to compensate for blood flow, perfusion, and oxygen delivery through sympathetic activity stimulation and activation of the renin-angiotensin system. This compensatory response leads to intra-renal vasoconstriction. Although hemodynamic normalization through resuscitation can restore blood pressure and alleviate vasoconstriction, it cannot completely halt the injury process, particularly ischemic reperfusion injury (5).

A decrease in total protein (TP) levels following hemorrhagic shock is attributed to mechanical effects, such as the direct loss of protein molecules with the shed blood and the potential leakage of proteins into the interstitial space. This leakage may result from increased capillary membrane permeability and endothelial glycocalyx damage secondary to shock-induced stress (37).

Therapeutic effects on the liver and kidney

Based on blood chemistry and histopathological comparisons, it can be concluded that UST therapy provides protective effects only in groups combined with FT or Dx. UST therapy without FT does not yield good protective results. The influence of FT in resuscitation is evident through decreased blood chemistry values of ALP and ALT, although AST variable values remain elevated, to a small degree. Similarly, the values of TP, BUN, and Cr indicate that all treatments incorporating FT have higher protective scores compared to UST+Dx and UST alone. Based on histopathological observations, UST combined with FT or Dx consistently shows higher protective rankings than standalone UST.

The use of intravenous fluid therapy to manage hemorrhagic shock (HS) has long been practiced to restore physiological parameters (38). Fluid resuscitation during the "golden hour" is critical to maintaining organ function and preventing mortality (39,40). Post-HS fluid resuscitation is an essential therapeutic approach to enhance intravascular volume and organ perfusion in critically injured patients (6). However, depending on its composition and volume, fluid therapy is also reported to have adverse effects on organ function (5).

Fluid therapy helps improve organ perfusion but does not fully protect the liver from damage. Degeneration due to hypoperfusion may be mitigated, but hepatocyte apoptosis can worsen during reperfusion due to increased ROS production, inducible Nitric Oxide Synthase (iNOS) induction, and calcium homeostasis disruption (32,41). The use of lactated Ringer’s may reduce bcl-2 expression and hepatic ATP reserves while increasing apoptosis, possibly due to the D-lactate content, which influences immune response and macrophage polarization (42,43). In the kidneys, crystalloids easily shift into interstitial spaces, leading to hemodilution and glycocalyx damage, which elevate creatinine and BUN levels (44,45). Although not entirely preventing injury, FT aids metabolic recovery and reduces acidosis (46), which contributes to inflammation and vascular dysfunction (47).

The expectation for UST use in hemorrhagic shock management is to prevent the development of post-HS multi-organ dysfunction. Its proposed mechanism of action involves suppressing inflammation through stimulated vagus nerve activity. It is anticipated that by reducing inflammation, circulating oxidative stress will also decrease, thereby allowing liver enzyme levels and kidney filtration capacity to return to normal (31,48). A study has demonstrated its inhibitory effects on TNF-α release and splenic macrophages. This indicates that afferent (sensory) signals from the vagus nerve influence efferent (motor) signals in the form of inflammatory reflexes, which contribute to the regulation of innate immune responses or cytokine homeostasis (49). The inflammatory reflex is defined as a physiological signaling mechanism in which afferent vagus nerve signals, activated by cytokines or pathogen-derived products, trigger efferent vagus nerve responses to regulate pro-inflammatory cytokine production (50). Vagus nerve stimulation can attenuate inflammatory responses by activating neuroimmune circuits known as the cholinergic anti-inflammatory pathway (50).

Based on this study’s findings, it can be stated that the UST used likely has effects on suppressing inflammatory mediators. However, its application as a standalone therapy without FT needs reevaluation. The mechanism of UST leading to protective effects on the liver and kidneys post-hemorrhagic shock likely involves neuromodulation through three mechanisms: thermal, cavitation, and acoustic radiation (19). The thermal effect occurs when acoustic energy from ultrasound penetrates tissue and induces vibrations. These vibrations are then converted into heat (51). Neurons are highly sensitive to temperature changes; even small increases (<1°C) can alter action potential kinetics and ion channel activity (52). Various temperature-sensitive transient receptor potential (TRP) channels, such as TRPV1 and TRPV4, exist in neuronal membranes. These receptors can even be activated at 37°C (53). Specifically, slight, moderate, or high-temperature increases open calcium (Ca) channels, leading to intracellular Ca2+ influx through TRP channels (53).

The second mechanism is cavitation. Cavitation is a non-thermal ultrasound therapy mechanism that utilizes sound wave-induced vibrations to create spaces and bubbles within tissues. These cavities oscillate in response to acoustic waves, producing acoustic emissions, radiation, and flow, which can cause biological effects (53). This phenomenon deforms the lipid bilayer membrane, ultimately triggering action potentials and enhancing synaptic transmission (54).

The final mechanism involves acoustic radiation pressure, also known as mechanical effects. The mechanical force produced by stable acoustic pressure on target neurons stretches the cell membrane, causing conformational and structural changes in mechanosensitive ion channels on the cell membrane (55). This process enables these ion channels to respond to mechanical stimuli, contributing to the modulation of cellular activity and nerve function (53). Exposure to ultrasound with an intensity of 1 W/cm² and continuous waves can increase the amplitude of compound action potentials (CAP) (56). CAP is a signal recorded from nerve trunks consisting of many axons. The increased CAP amplitude by ultrasound is due to non-thermal effects (mechanical stimulation) for two reasons. The first is that the stretch-activated ion channels are much more sensitive to mechanical than thermal stimulation. The more ion channels open, the more Na+ and K+ ions move in and out of the cell membrane, thereby increasing CAP amplitude. The second reason is that increased temperature does not enhance CAP amplitude. This suggests that the thermal effects of ultrasound can partially deactivate ion channels, allowing fewer ions to pass through the membrane, thus reducing CAP amplitude. This is evidenced by increasing intensity (2 and 3 W/cm²), which decreases CAP amplitude (56).

The difference between UST combined therapy and UST without FT shows no significance. This is likely due to the suboptimal intensity of the ultrasound used. The UST therapy applied was 10 minutes per session with a 30-minute interval between applications, at a frequency of 1 MHz, intensity of 1 W/cm², and five applications in total. The frequency and intensity applied were lower than those used in previous studies (18). This aimed to determine the potential of non-invasive UST for vagus nerve stimulation. Low-intensity continuous ultrasound therapy (LICUS) is sufficient for thermal induction to produce nerve regulation and thrombolysis effects. In this study, we did not examine the changes in the vagus nerve, either in terms of anatomy, microanatomy, or neurotransmitter levels. Further research is needed to investigate changes in inflammatory mediator levels in serum and organs through immunohistochemistry, which may help clarify the cellular mechanisms underlying the protective effects of UST via vagus nerve stimulation pathways.

Conclusion

Ultrasound therapy in combination tends to show better protective effects on the liver and kidneys compared to using ultrasound therapy alone. It is necessary to test both single and combination ultrasound therapies with higher intensities and a larger sample size to determine the optimal protective effects against organ damage.

Acknowledgment               

The author is grateful to the Veterinary Teaching Hospital, Division of Surgery and Radiology, Division of Pathology, School of Veterinary Medicine and Biomedical Science, IPB University for providing the location for this research. The authors also express their gratitude for the financial assistance received from the Center for Higher Education Funding (BPPT) and the Indonesia Endowment Fund for Education (LPDP), as acknowledged in decree 01802/J5.2.3/BPI.06/9/2022.

Conflict of interest

There is no conflict of interest.