Organ damage after hemorrhagic shock: roles of anesthesia, shock duration, and assessment time

OLIVEIRA, FILIPE RODOLFO M.B.; RAMOS, HANNA P.; MARIOT, LUANA N.; FERNANDES, DANIEL; ASSREUY, JAMIL; SORDI, REGINA

doi:10.1590/0001-3765202620251220

Abstract

Hemorrhagic shock (HS) is a condition associated with organ failure. Animal models are widely used to study pathophysiology and potential therapies. Our aim was to clarify how anesthetic choice, shock duration, decompensation phase length, and time post-reperfusion influence plasma damage markers in HS. For this, male rats were anesthetized with either thiopental or ketamine/xylazine and subjected to HS. Blood pressure was reduced to 40 mmHg for 60 or 90 minutes, followed by reperfusion with shed blood. Blood samples were collected 4 or 24 hours after reperfusion. Liver and renal damage were observed in animals that underwent a 90-minute shock, but not a 60-minute shock. Animals anesthetized with ketamine/xylazine exhibited higher hepatic aminotransferases levels than those anesthetized with thiopental, suggesting more severe liver injury in the former group. AST levels were higher 4 hours after reperfusion. The duration of decompensation phase was positively correlated with liver injury but not with kidney dysfunction. Cardiovascular parameters and other organ damage markers were similar among the groups. Therefore, HS protocols in rats require a minimum of 90 minutes of the ischemic phase to induce consistent organ dysfunction detectable through plasma markers. The duration of the decompensation phase directly influences the outcome.

Key words
animal model; hemorrhage; hypovolemic shock; liver injury; organ failure; trauma

INTRODUCTION

Hemorrhagic shock (HS) is a condition commonly associated with trauma, which is one of the leading causes of death among individuals up to 45 years old (Eastridge et al. 2019). HS is characterized by severe hypovolemia, which leads to a decrease in tissue perfusion and, potentially, can induce multiple organ failure (MOF) (Denk et al. 2018). The resuscitation/reperfusion after severe hemorrhage, although necessary for volume reposition, not only improves tissue perfusion but also aggravates injury and triggers a systemic inflammatory response, also contributing to the development of MOF (Eltzschig & Eckle 2011). In addition, several factors contribute to an elevated risk of HS-associated MOF, including sex, age, and underlying comorbidities (Trentzsch et al. 2014, Vanzant et al. 2015). Among those who survive HS, the occurrence of MOF varies widely from 7% to 58.5% (Fröhlich et al. 2014, Hutchings et al. 2017, Minei et al. 2012, Morrison et al. 2013, Probst et al. 2009).

HS presents two phases: the first is the compensatory phase, during which the body activates several self-regulatory mechanisms to maintain perfusion pressure and blood flow to vital organs such as brain and heart, even if it results in harm to other organs (e.g., intestine and liver) (Libert et al. 2016). Among the compensatory mechanisms are the activation of neurohumoral systems and an increase in catecholamine release, leading to vasoconstriction and increased heart rate. If the hemorrhage remains uncontrolled, the compensatory mechanisms become depleted, leading to myocardial depression and progressive vasodilation. This characterizes the second phase, known as decompensation (Thiemermann et al. 1993, Krizbai et al. 2005).

Experimental HS protocols have been utilized for many years, and the continuous interest surrounding this condition has led to a wide variety of methods. A review paper has pointed out that this lack of consistency impacts the comparability of studies (Tremoleda et al. 2017), thereby hindering progress in this field of research. For instance, in experimental models with recovery, there is no consensus regarding fundamental variables such as the target blood pressure during shock, the duration of the hemorrhage period, or the choice of the anesthetic agents.

Given the wide range of experimental methods used to study HS, combined with the limited information available about the impact of certain variables on the results, mainly in long term analyses, the hypothesis of this study is that the type of anesthetics and the time spent in decompensation phase influence the organ injury and dysfunction in a rat model of HS. Therefore, this study aimed to investigate, in a rat model of HS, the impact of the shock length, decompensation phase duration, and anesthetic choice on quickly measurable parameters that are widely used to quantify HS-induced organ failure.

MATERIALS AND METHODS

Animals and Ethics Committee Approval

The animal protocols used in this study were previously approved by the Ethics Committees on Animal Use of both the Federal University of Santa Catarina (no. 7396250219) and the State University of Ponta Grossa (no. 048/2015), in accordance with the ARRIVE (Animal research: reporting in vivo studies) guidelines for animal experimentation (Kilkenny et al. 2010).

Fifty-two male Wistar rats (Rattus norvegicus) aged 2 months and weighing between 250 and 350 g were obtained from the animal facilities of the institutions. They were housed in cages with capacity for 5 rats (45 x 34 x 16 cm) in a ventilated rack and kept under controlled conditions of temperature (21 ± 1 °C), luminosity (12-hour light-dark cycle; lights on at 7 a.m.), with food and water provided ad libitum. Following a 30-minute acclimatization period in the experimental facility, animals from different cages were individually identified and given a unique number. Then, the animals were randomly assigned, using their number, to form experimental groups (details of which are provided in each section). The experimental HS procedure commenced at 8 a.m. and varied in duration according to the assigned protocol for each animal. After the procedure, the animals were kept in heated cages until fully recovered. Each animal served as an individual experimental unit, and the following humanitarian endpoints were considered: weight reduction of ≥ 20% compared to initial weight, hypothermia (4°C below the temperature of the control group), severe dehydration (assessed by pitching of the skin in the back of the head of the animals), or when the experimenter judged that animal suffering exceeded that expected for the procedure (such as prolonged prostration and intense respiratory distress). No animals were sacrificed for these reasons.

Hemorrhagic shock and cardiovascular parameters

The HS model was performed as previously described with some modifications (Sordi et al. 2016, 2017). Briefly, after anesthesia, as described in the experimental groups below, the animals had their left femoral artery and vein cannulated using polythene tubing (PE25; Smiths Medical International Ltd, Kent, UK). Body temperature was monitored using an ear thermometer and maintained at 37 ± 0.5°C using a heating mat during the surgery. Arterial access was used for both blood pressure recording and blood withdrawal. Mean arterial pressure (MAP) was recorded by a transducer coupled to a PowerLab 8/30 (AD Instruments Pty Ltd., Castle Hill, Australia) running the proprietary software LabChart 8®. After MAP had stabilized (MAP = 100.6 ± 12.54 and 101.8 ± 13.37 mmHg for animals anesthetized with ketamine/xylazine and thiopental, respectively), blood was withdrawn in a rate of 1 ml/minute to achieve a target MAP of 40 ± 2 mmHg. The withdrawn blood (average volume = 9.86 ± 3.9 and 9.1 ± 1.5 mL per animal for the ketamine/xylazine and thiopental groups, respectively) was stored in a refrigerator and mixed with 0.2 ml of heparin (100 IU/ml) to prevent clotting. To maintain MAP at the desired values, additional blood withdrawal or administration of shed blood through the femoral vein was performed during the compensatory or decompensatory phases, respectively. After 60 (HS 60) or 90 (HS 90) minutes, the remaining shed blood warmed at 24 ± 2°C, was re-infused in a 5-minute period, followed by the injection of 1.5 ml/kg of Ringer-lactate (i.v.). Animals were euthanized 4 or 24 hours after reperfusion (see Time of analysis). Animals euthanized 4 hours after the reperfusion were kept under anesthesia throughout the entire period. Animals euthanized 24 hours after the reperfusion had their cannulas removed, vessels ligated, and skin sutured. Then, they were left in warm cages to recover with free access to water and food. Sham-operated rats were used as control and underwent identical surgical procedures, without hemorrhage and reperfusion. Four or 24 hours later, anesthetized animals had blood samples collected with tubes containing separation gel (Tubo Gel BD SST® II Advance®, Curitiba, Brazil). Blood was centrifuged to obtain plasma (2200 g, 10 minutes) and each sample was stored in a numbered microtube corresponding to a specific animal. Creatinine, urea, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine phosphokinase (CPK), alkaline phosphatase (ALP), and amylase (AMY) were determined according to the manufacturer’s instructions (Siemens Healthineers, Erlangen, BY, Germany). All assays were performed by an analyst blinded to the experimental group allocation.

Variables analyzed in liver injury and kidney dysfunction

Shock duration

To investigate the impact of shock duration on organ damage, the animals were anesthetized with ketamine and xylazine (100 and 10 mg/kg, respectively, i.m.) and maintained using 30 mg/kg of ketamine, i.p., as necessary. They were then subjected to 60 (HS 60) or 90 minutes (HS 90) of shock. Sham-operated animals were used as controls. Tramadol (10 mg/kg, i.p.) was administered as analgesic every 12 hours. All animals were euthanized 24 hours after surgery.

Anesthetic

To compare the influence of different anesthetics, two different protocols were used: sodium thiopental (100 mg/kg, i.p., maintained using 10 mg/kg, i.v.) or ketamine and xylazine (as described before). Animals were subjected to 90 minutes of shock. Tramadol (10 mg/kg, i.p.) was used as analgesic every 12 hours. Animals were evaluated 24 hours after surgery.

Time of analysis

The animals subjected to 90 minutes of shock were evaluated 4 (acute model) and 24 hours (recovery model) after the surgery. These points were selected to capture both the acute and the more prolonged phases of the post-ischemic response. This study was conducted using thiopental as anesthetic, as described above.

Time spent in the decompensation phase

The decompensation duration was defined as the time from when the animal needed blood reinfusion to maintain PAM at 40 ± 2 mmHg until the onset of reperfusion (which occurred either 60 or 90 minutes after the hemorrhage, depending on the experimental group). Thus, the duration of the decompensation phase varied among the animals.

To verify if there was a correlation between decompensatory phase duration and organ damage severity, data from rats anaesthetized with ketamine and xylazine were used to compute Spearman’s correlation test.

Reagents

Unless otherwise stated, all compounds were from Sigma-Aldrich Company Ltd (Poole, Dorset, UK). Ketamine and xylazine were purchased from Ceva (Paulínia, SP, Brazil); heparin was from Blausiegel (Cotia, SP, Brazil); tramadol was from Teuto (Anápolis, GO, Brazil); sodium thiopentone (Thiopentax) from Cristalia (Itapira, SP, Brazil); Ringer-lactate was from Eurofarma (Ribeirão Preto, SP, Brazil).

Statistical analysis

The results are expressed as dot plots with bars and mean ± SD are shown. Based on SD and difference of means of blood pressure from previous studies (Sordi et al. 2017, 2016) a sample size of 7 animals in each group was calculated to detect difference with a power of 80% and α = 0.05 using a two-tailed test. However, due to the expected mortality in HS models, we planned for 12 animals in each group. For this reason, the n of animal ranges from 7 to 12 as indicated in the figures. All animals that survived to the procedures were included in the study. The Shapiro-Wilk test was used to assess the distribution of data, and the homogeneity of variances was verified by the Bartlett test. When necessary, values were transformed into logarithmic values to achieve normality and homogeneity of variances. To analyze the difference between two groups in the same experimental context (i.e., same anesthetics and time of analysis), the Student’s t-test was employed. One-way analysis of variance (ANOVA) followed by the Tukey post-hoc test was carried out for analysis comparing two or more groups in different contexts. Spearman correlations were performed to examine the strength of associations in non-normally distributed samples. A p value of less than 0.05 was considered significant. Data were analyzed using GraphPad Prism® 9.0 software (California, USA).

RESULTS

Role of shock duration in plasma markers of organ damage

Twenty-four hours after the reperfusion, the animals that experienced 90 minutes of shock showed significant elevations in plasma AST and ALT levels compared with sham-operated rats (Fig. 1a and b). Urea levels (Fig. 1c) were slightly elevated in HS 90 group, but not creatinine (Fig. 1d). In contrast, no changes in tissue markers were found in HS 60 group.

Figure 1
Duration of shock interferes with liver injury and kidney dysfunction. Rats were anesthetized with ketamine/xylazine (100 and 10 mg/kg, respectively) and subjected to 60 (HS 60) or 90 minutes (HS 90) of shock. Blood was obtained 24 hours after reperfusion. Sham-operated rats were used as control. a) aspartate aminotransferase (AST); b) alanine aminotransferase (ALT); c) urea; d) creatinine. Each symbol represents one animal, vertical bars represent mean and error bars represent SD (n = 7 – 9). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test (*p < 0.05 vs. sham-operated rats).

Role of anesthetics in plasma markers of organ damage

When compared with their respective sham-operated groups, rats subjected to HS 90 and euthanized 24 hours after surgery exhibited elevated levels of markers of liver injury and kidney dysfunction. Notably, the choice of anesthetic had a substantial impact on these markers. Animals anesthetized with ketamine/xylazine presented levels of liver injury markers ~4-5-fold higher than those anesthetized with thiopental (Fig. 2a and b). This pattern was even observed for AST levels when comparing the sham-operated rats.

Figure 2
Anesthetic agents interfere with liver injury, but not with kidney dysfunction. Rats were anesthetized with thiopental (100 mg/kg; open symbols) or ketamine/xylazine (100 and 10 mg/kg, respectively; closed symbols) and were subjected to the 90-minute shock protocol (HS 90). Blood was obtained 24 hours after reperfusion. Sham-operated rats were used as control. a) aspartate aminotransferase (AST); b) alanine aminotransferase (ALT); c) urea; d) creatinine. Each symbol represents one animal, vertical bars represent mean and error bars represent SD (n = 7 – 9). Statistical analysis was performed using t test to evaluate the difference between sham-operated rats and HS with the same anesthetic scheme. One-way ANOVA followed by Tukey’s post hoc test was used for the other comparisons (*p < 0.05).

Regarding kidney dysfunction, both HS 90 groups showed a significant increase in urea (Fig. 2c) and creatinine (Fig. 2d) when compared to their controls. In this case, the values observed with both anesthetics were similar. Notably, although the pairwise comparison between the two groups (creatinine levels using ketamine/xylazine) using a t-test indicated a statistically significant difference (Fig. 2d), this effect was not retained when a third group was included in the analysis using one-way ANOVA (Fig. 1d). This change likely reflects the increased variability introduced by the additional group and the more conservative nature of ANOVA when adjusting for multiple group comparisons.

Acute versus recovery model in plasma markers of organ damage

Because lower levels of organ damage markers were observed in animals anesthetized with thiopental, we investigated the time course of liver injury and kidney dysfunction markers (Fig. 3). Overall, markers were elevated 4 hours after shock and remained elevated at 24-hour time point, except for ALT, which showed a reduction 24 hours later.

Figure 3
Comparison of acute and recovery model. Rats were anesthetized with thiopental (100 mg/kg) and were subjected to 90-minutes shock protocol (HS 90). Blood was obtained 4 hours (acute) or 24 hours (recovery) after reperfusion. Sham-operated rats were used as control. a) aspartate aminotransferase (AST); b) alanine aminotransferase (ALT); c) urea; d) creatinine. Each symbol represents one animal, vertical bars represent mean and error bars represent SD (n = 7 – 9). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test (*p < 0.05).

Role of decompensation phase in plasma markers of organ damage

The duration of the decompensatory phases of HS was 9.3 ± 6.7 and 33.4 ± 16.3 minutes for HS60 and HS90 groups, respectively. The general duration of the decompensatory phase (HS60 and HS90) was 24.1 ± 17.8 minutes. A significant positive correlation (p ≤ 0.001) was observed between the time spent in decompensation and AST (Fig. 4a) and ALT (Fig. 4b) levels. On the other hand, kidney dysfunction markers were not significantly correlated to the time in the decompensatory phase (Fig. 4c e 4d).

Figure 4
Duration of decompensation phase is directly related to liver injury. Rats were anesthetized with ketamine/xylazine (100 and 10 mg/kg, respectively) and were subjected to HS protocol. Blood was obtained 24 hours after reperfusion. Values of a) aspartate aminotransferase (AST); b) alanine aminotransferase (ALT); c) urea, and d) creatinine were plotted on the Y axis and the time (minutes) that the animals spent in the decompensation phase was plotted on the X axis. Each symbol represents one animal. Spearman correlation test was computed. The exact rho and p values are shown in figures.

Cardiovascular parameters

Figure 5 shows the MAP curves during the HS procedure in animals anesthetized with ketamine/xylazine (Fig. 5a) and thiopental (Fig. 5b), respectively. After stabilization of MAP, which generally occurred between 10 and 20 minutes following vessel cannulation, bleeding was initiated, and approximately 5 to 10 minutes later the target MAP was reached (hemorrhage phase). After 90 minutes, animals underwent reperfusion. The values shown at the “post-reperfusion” time point were collected 20 minutes after the end of reperfusion, as this was the point at which MAP stabilized. A statistically significant difference in MAP was observed in animals subjected to HS compared with sham-operated animals one hour after reperfusion in both groups. This difference remained in the acute (4 hours) assessment for both animals anesthetized with ketamine/xylazine (Fig. 5c) and those anesthetized with thiopental (Fig. 5d). However, 24 hours later, MAP values returned to levels comparable to those in sham-operated animals in both groups. Regarding heart rate, animals anesthetized with ketamine/xylazine exhibited bradycardia 24 hours after HS (Fig. 5e), which was not observed in the thiopental-anesthetized group (Fig. 5f). Sham-operated animals evaluated at 4- and 24-hour time points were grouped together, as no differences were found between them (panels c–f).

Figure 5
Cardiovascular parameters in anesthetized rats subjected to HS. Rats were anesthetized with ketamine/xylazine (100 and 10 mg/kg, respectively; closed symbols) or thiopental (100 mg/kg; open symbols) and were subjected to 90-minute shock protocol. MAP was recorded during the HS procedure (panels a and b). MAP (panels c and d) and HR (panels e and f) were also recorded 4 hours and 24 hours after the HS surgery. For graphs a and b, each symbol represents mean and error bars represent SD (n = 6 – 9). Statistical analysis was performed using two-way ANOVA followed by Sidak’s test to evaluate the differences in MAP between sham-operated and HS-operated groups at the different timepoints (****p < 0.0001 and ***p < 0.001). For graphs c – f, each symbol represents one animal, bars represent mean, and error bars represent SD (n = 7 – 9). One-way ANOVA followed by Tukey’s post hoc test was used for comparisons (**p < 0.01 and *p < 0.05).

Secondary organ damage markers

Additional markers of organ damage across different systems were also quantified (Fig. 6). Animals anesthetized with either ketamine/xylazine or thiopental showed elevated creatine phosphokinase (CPK) levels 4 h after hemorrhagic shock (Fig. 6a and 6b). This increase persisted up to 24 h in animals anesthetized with thiopental. Alkaline phosphatase (ALP) levels were also transiently elevated 4 h after the insult in the ketamine/xylazine group (Fig. 6c), which was not observed in thiopental-anesthetized animals (Fig. 6d). Amylase (AMY - Fig. 6e and 6f) levels did not show significant changes following hemorrhagic shock.

Figure 6
Secondary organ damage markers. Rats were anesthetized with ketamine/xylazine (100 and 10 mg/kg, respectively; closed symbols, panels a, c, and e) or thiopental (100 mg/kg; open symbols, panels b, d, and f) and were subjected to the 90-minute shock protocol (HS 90). Blood was obtained 4 or 24 hours after reperfusion. Sham-operated rats were used as control. a and b) creatinine phosphokinase (CPK); c and d) alkaline phosphatase (ALP); e and f) amylase (AMY). Each symbol represents one animal, vertical bars represent mean and error bars represent SD (n = 7 – 12). Statistical analysis was performed using one-way ANOVA followed by Tukey’s test (**p < 0.01 and *p < 0.05).

DISCUSSION

The main finding of the present study is that the choice of anesthetic, the duration of shock, and the duration of the decompensatory phase influence the degree of organ injury and dysfunction in a rat model of HS. By correlating the duration of the decompensation phase for each animal with plasma marker levels, we have shown that decompensatory phase highly impacts liver injury parameters, with minimum effect on kidney dysfunction.

Although several parameters have been extensively discussed in an interesting review about models of HS (Tremoleda et al. 2017), to the best of our knowledge, this is the first study to systematically compare and examine the importance of time spent in decompensation phase for organ injury in the HS model in rats. The findings provide valuable insights into the factors that contribute to the variability in the HS model and offer guidance for researchers in the design and interpretation of their experiments.

Animal models have proven to be valuable tools for understanding the HS pathophysiological mechanisms and for exploring potential new therapies. Among the various models used to study the effects of ischemia-reperfusion injuries on tissues, the HS in rats is one of the most employed (Fülöp et al. 2013). In addition, despite the widespread use of animal models of HS, there is a lack of standardized protocols, with a multitude of variables reported in the literature (Fülöp et al. 2013) . Therefore, to better understand the key parameters that contribute to the development of organ injury in the HS model, we conducted a comparative study. Our investigation involved two types of anesthetics regimens, two different shock durations, and two post-reperfusion analysis times. By comparing the impact of these variables on organ injury and dysfunction in the HS model, we aimed to identify the most influential factors within the HS model and our findings, ultimately, provide important insights for future research using this model.

The studies employing HS model adopt target MAP values ranging from 30 (Dunberry-Poissant et al. 2018, Lin et al. 2020, Mann et al. 1997) to 40 mmHg (Proctor et al. 2015, Lei et al. 2015, Liu et al. 2011, Wu et al. 2008, Zhang et al. 2019, Patel et al. 2022, 2023). As our main objective was investigating recovery model, to avoid excessive mortality, we opted for 40 mmHg as our starting point. Next, we investigated whether the duration of the shock would impact on organ failure. Interestingly, when evaluated 24 hours after shock recovery, animals that underwent a 60-minute shock did not exhibit significant increases in AST and ALT levels, indicating no liver injury. In contrast, all the animals that experienced a 90-minute shock demonstrated increased levels of these markers, indicating some degree of liver injury. The gastrointestinal tract is particularly sensitive to damage caused by trauma and ischemia (Wrba et al. 2017). Ischemia-induced damage to the gut-blood barrier can lead to bacterial translocation and endotoxins release from intestinal lumen into the liver via the portal vein. This can trigger the production and release of several proinflammatory local mediators, which can cause a significant decrease of hepatic microcirculation and hepatocyte function (Deitch et al. 1990, Swank & Deitch 1996, Koo & Liang 1977). Our data suggest that to observe a consistent increase in AST and ALT levels, an ischemic period of at least 90 minutes is required to ensure the progression to the decompensatory phase of shock.

Regarding renal function, animals that underwent a 90-minute procedure demonstrated an increase in urea and creatinine levels 4 hours and 24 hours after reperfusion, while no changes were observed in animals subjected to the 60-minute protocol. Although acute kidney injury is associated with higher mortality in post-trauma patients (Coca et al. 2012, Oliveira et al. 2022), observing these damages in an experimental model in rodents can be a challenge. Previous studies have shown that renal cells injury appeared to be sublethal, with animal urine composition returning to normal 48 hours after ischemia (Kreisberg et al. 1976). Hence, it can be concluded that, similar to the liver, a minimum of 90 minutes of HS is required to observe some degree of kidney dysfunction.

Interestingly, the choice of anesthetic had a significant impact on hepatic damage markers, but not on other parameters. After 90 minutes of shock, all animals exhibited renal and hepatic injuries; however, rats anesthetized with ketamine and xylazine showed elevated levels of AST and ALT in comparison to those anesthetized with thiopental. Although several anesthetic regimens have been reported in the literature, combinations containing ketamine remain the most common choice in rodent experimental models, administered via intramuscular, intravenous, or intraperitoneal routes (Veilleux-Lemieux et al. 2013). Previous studies have reported hepatocellular toxicity and histological changes in rodents that received ketamine alone or in combinations (Kalkan et al. 2014, Thompson et al. 2002), which may partially explain the greater hepatic damage and the statistically higher AST levels observed even in sham-operated groups anesthetized with ketamine/xylazine compared with thiopental. Indeed, serum AST levels have been shown to increase from 3 to 48 hours following ketamine/xylazine injection (Chen et al. 2020). Moreover, ketamine can induce mitochondrial dysfunction and apoptosis in hepatocytes, leading to liver cell damage and enzyme leakage. In contrast, thiopental, a barbiturate used for anesthesia, typically does not cause significant increase in hepatic enzymes (Thakkar & Wu 2025).

Thiopental potentiates GABA-A-mediated inhibitory neurotransmission, resulting in central nervous system depression and potential decreases in blood pressure, as it may reduce the amount of endogenous catecholamines (Aksoy et al. 2015, Kavanagh et al. 1991). In contrast, ketamine, an NMDA receptor antagonist, enhances catecholamine release and increases sympathetic tone (Aksoy et al. 2014). Xylazine, an α₂-adrenergic agonist, produces sedation, analgesia, bradycardia, and hypotension through central inhibition of norepinephrine release. However, because no major differences in MAP, HR, or other multiple-organ damage markers were observed between animals anesthetized with thiopental and those receiving ketamine/xylazine, the differences in organ injury cannot be attributed solely to the cardiovascular effects of these anesthetic agents.

Additionally, we compared the differences between 4 and 24 hours after HS surgery in animals anesthetized with thiopental. These time points were selected to capture both the acute phase, characterized by acute inflammatory and hemodynamic changes (Sordi et al. 2015, 2016), and the more prolonged phases of the post-ischemic response, during which secondary inflammatory, metabolic, and tissue repair processes become more prominent (Sordi et al. 2024). Interestingly, liver damage was slightly more pronounced at 4 than 24-hour time point. This may be due, at least in part, to the animals recovering from the initial insult after 24 hours. Previous studies have shown that there are differences in the mediators that contribute to the early and later stages of shock. For instance, enzymes such as inducible and neuronal nitric oxide synthase (iNOS and nNOS, respectively), as well as cyclooxygenase-2 (COX-2), demonstrate increased activity primarily in the initial phase of shock, which is strongly associated with subsequent organ failure (Hierholzer & Billiar 2001, Sordi et al. 2016). However, the precise role that these mediators play in later stages of shock is still not well understood, as studies evaluating long-term follow-ups are scarce.

Moreover, we investigated whether the duration of the decompensatory phase affected the organ damage markers. The decompensatory phase refers to the period during which blood or fluids are administered to the animal to maintain MAP at desired values after regulatory mechanisms begin to fail. Remarkably, the correlations analysis revealed a strong and positive correlation between liver injury markers and the duration of the decompensation phase, which was not observed for kidney dysfunction. As discussed earlier, kidneys appear to have a more efficient repair mechanism (Oliveira et al. 2024), while livers are more vulnerable to ischemic damage. Interestingly, the rise in kidney dysfunction markers is less common than the elevation of injury markers from other systems in individuals experiencing multiorgan dysfunction following HS (Hutchings et al. 2017). In addition, although the acute kidney injury is one of the most fatal post-HS complications, its incidence remains relatively low, affecting only up to 10% of trauma patients (Martínez et al. 2013, Awad et al. 2009, Bagshaw 2006, Hoste & Schurgers 2008).

This study presents some limitations that should be considered when interpreting the results. Only young male rats were used in the experiments, which may limit the generalization of findings to other populations, including different age groups and female animals. Very minimal traumatic injury was induced in our model, as our focus was the investigation of specific parameters rather the trauma itself. This approach contrasts with trauma-induced HS in clinical scenario, where the nature of trauma significantly affects the outcome. In the acute model, animals euthanized 4 hours after the insult were kept anesthetized throughout the experiment, which could have affected the results. We were not able to conduct analyses beyond 24 hours after HS, however, a previous study showed that no differences were observed between 24 hours and 72 hours creatinine levels in HS animals (Dunberry-Poissant et al. 2018). Histological analysis of kidney and liver tissues were not performed as our main objective was to investigate the parameters in the model that contribute to consistent increases in plasma markers of organ damage, as these parameters are used more often as quick assessment in clinical practice. Another limitation of the present study is that isoflurane or other inhalational anesthetics were not available at our facility at the time of the experiments; therefore, their potential differential effects on organ injury could not be assessed. Future studies incorporating volatile anesthetics such as isoflurane may help to further delineate the influence of anesthetic choice on the observed outcomes. Lastly, we used heparin to prevent the formation of clots, which has various potential beneficial effects beyond the anticoagulation (Tyrrell et al. 1999). Indeed, heparin has been shown to exert anti-inflammatory and endothelial-protective actions, reducing leukocyte adhesion, microvascular thrombosis, and tissue injury (Li et al. 2015, Potje et al. 2021, Stark 2021). Therefore, our results should be cautiously interpreted, as these pleiotropic effects might attenuate some of the pathophysiological responses associated with hemorrhagic shock.

In conclusion, this study shows that: a) to induce increase in serum markers of liver injury and kidney dysfunction through HS model in rats, minimal shock duration of 90 minutes is required; b) for most of the parameters assessed here, the choice of anesthetic did not significantly affect the outcome. However, thiopental appears to be the most appropriate anesthetic when evaluating hepatic-related parameters; c) liver injury appears to be slightly more severe in acute (4-hour) compared to the recovery model (24-hour); and d) the duration (> 30 minutes) of the decompensation phase is determinant for the intensity of liver injury, but not for kidney dysfunction.

Acknowledgements

The authors are grateful to the Multiuser Laboratory of Biology Studies at the Universidade Federal de Santa Catarina (LAMEB/UFSC) and to Hospital Universitário Polydoro Ernani São Thiago (HU-UFSC/EBSERH) for providing infrastructure for carrying out the experimental tests, and to Patrícia Casas and Letícia Kramer Pacheco for the technical assistance. Cristália Pharmaceutical Industries (São Paulo, Brazil) is also gratefully acknowledged for the gift of heparin. Fundings: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grant number 409018/2018-0; 405538/2021-9, 403615/2023-2, 409592/2024-2) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES Foundation), Brazil.

Data availability

Data will be made available on request.

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FRÖHLICH M, LEFERING R, PROBST C, PAFFRATH T, SCHNEIDER MM, MAEGELE M, SAKKA SG, BOUILLON B & WAFAISADE A. 2014. Epidemiology and risk factors of multiple-organ failure after multiple trauma. J Trauma Acute Care Surg 76: 921-928.
FÜLÖP A, TURÓCZI Z, GARBAISZ D, HARSÁNYI L & SZIJÁRTÓ A. 2013. Experimental models of hemorrhagic shock: a review. Eur Surg Res 50: 57-70.
HIERHOLZER C & BILLIAR TR. 2001. Molecular mechanisms in the early phase of hemorrhagic shock. Langenbecks Arch Surg 386: 302-308.
HOSTE EAJ & SCHURGERS M. 2008. Epidemiology of acute kidney injury: how big is the problem? Crit Care Med 36: S146-S151.
HUTCHINGS L, WATKINSON P, YOUNG JD & WILLETT K. 2017. Defining multiple organ failure after major trauma: a comparison of the Denver, Sequential Organ Failure Assessment and Marshall scoring systems. J Trauma Acute Care Surg 82: 534-541.
KALKAN Y, TOMAK Y, ALTUNER D, TUMKAYA L, BOSTAN H, YILMAZ A, UNAL D, KARA A & TURAN A. 2014. Hepatic effects of ketamine administration for 2 weeks in rats. Hum Exp Toxicol 33: 32-40.
KAVANAGH BP, RYAN MP & CUNNINGHAM AJ. 1991. Myocardial contractility and ischaemia in the isolated perfused rat heart with propofol and thiopentone. Can J Anaesth 38: 634-639.
KILKENNY C, BROWNE WJ, CUTHILL IC, EMERSON M & ALTMAN DG. 2010. The ARRIVE guidelines: animal research: reporting in vivo experiments. PLoS Biol 8: e1000412.
KOO A & LIANG IYS. 1977. Blood flow in hepatic sinusoids in experimental hemorrhagic shock in the rat. Microvasc Res 13: 315-325.
KREISBERG JI, BULGER RE, TRUMP BF & NAGLE RB. 1976. Effects of transient hypotension on the structure and function of rat kidney. Virchows Arch B Cell Pathol 22: 121-133.
KRIZBAI IA ET AL. 2005. Blood-brain barrier changes during compensated and decompensated hemorrhagic shock. Shock 24: 428-433.
LEI Y, PENG X, LIU L, DONG Z & LI T. 2015. Beneficial effect of cyclosporine A on traumatic hemorrhagic shock. J Surg Res 195: 529-540.
LI L, LING Y, HUANG M, YIN T, GOU S, ZHAN N, XIONG J, WU H, YANG Z & WANG C. 2015. Heparin inhibits the inflammatory response induced by LPS and HMGB1 by blocking the binding of HMGB1 to the surface of macrophages. Cytokine 72: 36-42.
LIBERT N, HARROIS A & DURANTEAU J. 2016. Haemodynamic coherence in haemorrhagic shock. Best Pract Res Clin Anaesthesiol 30: 429-435.
LIN B, GAO Y, LI Z, ZHANG Z, LIN X & GAO J. 2020. Cannabidiol alleviates hemorrhagic shock-induced neural apoptosis in rats by inducing autophagy through activation of the PI3K/AKT pathway. Fundam Clin Pharmacol 34: 640-649.
LIU F-C, LIU F-W & YU H-P. 2011. Ondansetron attenuates hepatic injury via p38 MAPK-dependent pathway in a rat haemorrhagic shock model. Resuscitation 82: 335-340.
MANN DV, ROBINSON MK, ROUNDS JD, DEROSA E, NILES DA, INGWALL JS, WILMORE DW & JACOBS DO. 1997. Superiority of blood over saline resuscitation from hemorrhagic shock: a 31P magnetic resonance spectroscopy study. Ann Surg 226: 653-661.
MARTÍNEZ RJÁ ET AL. 2013. Evolution and complications of chest trauma. Arch Bronconeumol 49: 177-180.
MINEI JP ET AL. 2012. The changing pattern and implications of multiple organ failure after blunt injury with hemorrhagic shock. Crit Care Med 40: 1129-1135.
MORRISON JJ, STANNARD A, RASMUSSEN TE, JANSEN JO, TAI NRM & MIDWINTER MJ. 2013. Injury pattern and mortality of noncompressible torso hemorrhage in UK combat casualties. J Trauma Acute Care Surg 75: S263-S268.
OLIVEIRA FRMB, ASSREUY J & SORDI R. 2022. The role of nitric oxide in sepsis-associated kidney injury. Biosci Rep 42: BSR20220093.
OLIVEIRA FRMB, SOUSA SOARES E, PILLMANN RAMOS H, LÄTTIG-TÜNNEMANN G, HARMS C, CIMAROSTI H & SORDI R. 2024. Renal protection after hemorrhagic shock in rats: possible involvement of SUMOylation. Biochem Pharmacol 227: 116425.
PATEL NM, OLIVEIRA FRMB, RAMOS HP, AIMARETTI E, ALVES GF, COLDEWEY SM, COLLINO M, SORDI R & THIEMERMANN C. 2023. Inhibition of Bruton’s tyrosine kinase activity attenuates hemorrhagic shock-induced multiple organ dysfunction in rats. Ann Surg 277: e624-e633.
PATEL NM ET AL. 2022. Inhibition of macrophage migration inhibitory factor activity attenuates haemorrhagic shock-induced multiple organ dysfunction in rats. Front Immunol 13: 886421.
POTJE SR ET AL. 2021. Heparin prevents in vitro glycocalyx shedding induced by plasma from COVID-19 patients. Life Sci 276: 119376.
PROBST C, PAPE H-C, HILDEBRAND F, REGEL G, MAHLKE L, GIANNOUDIS P, KRETTEK C & GROTZ MRW. 2009. 30 years of polytrauma care: an analysis of the change in strategies and results of 4849 cases treated at a single institution. Injury 40: 77-83.
PROCTOR JL, SCUTELLA D, PAN Y, VAUGHAN J, ROSENTHAL RE, PUCHE A & FISKUM G. 2015. Hyperoxic resuscitation improves survival but worsens neurologic outcome in a rat polytrauma model of traumatic brain injury plus hemorrhagic shock. J Trauma Acute Care Surg 79: S101-S109.
SORDI R, BOJKO L, OLIVEIRA FRMB, ROSALES TO, SOUZA CF, MORENO LW, ALVES GF, CARLOS J, VELLOSA R, FERNANDES D & GOMES JR. 2024. Doxycycline reduces liver and kidney injuries in a rat hemorrhagic shock model. Intensive Care Med Exp 12: 2.
SORDI R, CHIAZZA F, COLLINO M, ASSREUY J & THIEMERMANN C. 2016. Neuronal nitric oxide synthase is involved in vascular hyporeactivity and multiple organ dysfunction associated with hemorrhagic shock. Shock 45: 525-533.
SORDI R, CHIAZZA F, PATEL NSA, DOYLE RA, COLLINO M & THIEMERMANN C. 2015. ‘Preconditioning’ with low dose lipopolysaccharide aggravates the organ injury/dysfunction caused by hemorrhagic shock in rats. PLoS ONE 10: e0122096.
SORDI R, NANDRA KK, CHIAZZA F, JOHNSON FL, CABRERA CP, TORRANCE HD, YAMADA N, PATEL NSA, BARNES MR, BROHI K, COLLINO M & THIEMERMANN C. 2017. Artesunate protects against the organ injury and dysfunction induced by severe hemorrhage and resuscitation. Ann Surg 265: 408-417.
STARK K. 2021. Interplay between inflammation and thrombosis in cardiovascular pathology. Nat Rev Cardiol 18: 666-682.
SWANK GM & DEITCH EA. 1996. Role of the gut in multiple organ failure: bacterial translocation and permeability changes. World J Surg 20: 411-417.
THAKKAR B & WU GY. 2025. Ketamine hepatotoxicity: an underappreciated cause of liver damage - analysis by RUCAM. J Clin Transl Hepatol 13: 524-531.
THIEMERMANN C, SZABO C, MITCHELL JA & VANE JR. 1993. Vascular hyporeactivity to vasoconstrictor agents and hemodynamic decompensation in hemorrhagic shock is mediated by nitric oxide. Proc Natl Acad Sci USA 90: 267-271.
THOMPSON JS, BROWN SA, KHURDAYAN V, ZEYNALZADEDAN A, SULLIVAN PG & SCHEFF SW. 2002. Early effects of tribromoethanol, ketamine/xylazine, pentobarbital, and isoflurane anesthesia on hepatic and lymphoid tissue in ICR mice. Comp Med 52: 63-67.
TREMOLEDA JL, WATTS SA, REYNOLDS PS, THIEMERMANN C & BROHI K. 2017. Modeling acute traumatic hemorrhagic shock injury: challenges and guidelines for preclinical studies. Shock 48: 610-623.
TRENTZSCH H, NIENABER U, BEHNKE M, LEFERING R & PILTZ S. 2014. Female sex protects from organ failure and sepsis after major trauma haemorrhage. Injury 45: S20-S28.
TYRRELL DJ, HORNE AP, HOLME KR, PREUSS JMH & PAGE CP. 1999. Heparin in inflammation: potential therapeutic applications beyond anticoagulation. Adv Pharmacol 46: 151-208.
VANZANT EL ET AL. 2015. Advanced age is associated with worsened outcomes and a unique genomic response in severely injured patients with hemorrhagic shock. Crit Care 19: 77.
VEILLEUX-LEMIEUX D, CASTEL A, CARRIER D, BEAUDRY F & VACHON P. 2013. Pharmacokinetics of ketamine and xylazine in young and old Sprague-Dawley rats. J Am Assoc Lab Anim Sci 52: 567-570.
WRBA L, PALMER A, BRAUN CK & HUBER-LANG M. 2017. Evaluation of gut-blood barrier dysfunction in various models of trauma, hemorrhagic shock, and burn injury. J Trauma Acute Care Surg 83: 944-953.
WU R ET AL. 2008. Human vasoactive hormone adrenomedullin and its binding protein rescue experimental animals from shock. Peptides 29: 1223-1230.
ZHANG LM, ZHANG DX, FU L, LI Y, WANG XP, QI MM, LI CC, SONG PP, WANG XD & KONG XJ. 2019. Carbon monoxide-releasing molecule-3 protects against cortical pyroptosis induced by hemorrhagic shock and resuscitation via mitochondrial regulation. Free Radic Biol Med 141: 299-309.

Correspondence

Correspondence to: Regina Sordi Email: r.sordi@ufsc.br

Handling editor

Juliano Morimoto

Publication Dates

Publication in this collection
19 Oct 2026
Date of issue
2026

History

Received
12 Nov 2025
Accepted
11 May 2026

This is an open-access article distributed under the terms of the Creative Commons Attribution License

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[7] DEITCH EA, MORRISON J, BERG R & SPECIAN RD. 1990. Effect of hemorrhagic shock on bacterial translocation, intestinal morphology, and intestinal permeability in conventional and antibiotic-decontaminated rats. Crit Care Med 18: 529-536.

[8] DENK S ET AL. 2018. Role of hemorrhagic shock in experimental polytrauma. Shock 49: 154-163.

[9] DUNBERRY-POISSANT S, GILBERT K, BOUCHARD C, BARIL F, CARDINAL A-M, L’ECUYER S, HYLANDS M, LAMONTAGNE F, ROUSSEAU G & CHARBONNEY E. 2018. Fluid sparing and norepinephrine use in a rat model of resuscitated haemorrhagic shock: end-organ impact. Intensive Care Med Exp 6(1): 47.

[10] EASTRIDGE BJ, HOLCOMB JB & SHACKELFORD S. 2019. Outcomes of traumatic hemorrhagic shock and the epidemiology of preventable death from injury. Transfusion 59: 1423-1428.

[11] ELTZSCHIG HK & ECKLE T. 2011. Ischemia and reperfusion—from mechanism to translation. Nat Med 17: 1391-1401.

[12] FRÖHLICH M, LEFERING R, PROBST C, PAFFRATH T, SCHNEIDER MM, MAEGELE M, SAKKA SG, BOUILLON B & WAFAISADE A. 2014. Epidemiology and risk factors of multiple-organ failure after multiple trauma. J Trauma Acute Care Surg 76: 921-928.

[13] FÜLÖP A, TURÓCZI Z, GARBAISZ D, HARSÁNYI L & SZIJÁRTÓ A. 2013. Experimental models of hemorrhagic shock: a review. Eur Surg Res 50: 57-70.

[14] HIERHOLZER C & BILLIAR TR. 2001. Molecular mechanisms in the early phase of hemorrhagic shock. Langenbecks Arch Surg 386: 302-308.

[15] HOSTE EAJ & SCHURGERS M. 2008. Epidemiology of acute kidney injury: how big is the problem? Crit Care Med 36: S146-S151.

[16] HUTCHINGS L, WATKINSON P, YOUNG JD & WILLETT K. 2017. Defining multiple organ failure after major trauma: a comparison of the Denver, Sequential Organ Failure Assessment and Marshall scoring systems. J Trauma Acute Care Surg 82: 534-541.

[17] KALKAN Y, TOMAK Y, ALTUNER D, TUMKAYA L, BOSTAN H, YILMAZ A, UNAL D, KARA A & TURAN A. 2014. Hepatic effects of ketamine administration for 2 weeks in rats. Hum Exp Toxicol 33: 32-40.

[18] KAVANAGH BP, RYAN MP & CUNNINGHAM AJ. 1991. Myocardial contractility and ischaemia in the isolated perfused rat heart with propofol and thiopentone. Can J Anaesth 38: 634-639.

[19] KILKENNY C, BROWNE WJ, CUTHILL IC, EMERSON M & ALTMAN DG. 2010. The ARRIVE guidelines: animal research: reporting in vivo experiments. PLoS Biol 8: e1000412.

[20] KOO A & LIANG IYS. 1977. Blood flow in hepatic sinusoids in experimental hemorrhagic shock in the rat. Microvasc Res 13: 315-325.

[21] KREISBERG JI, BULGER RE, TRUMP BF & NAGLE RB. 1976. Effects of transient hypotension on the structure and function of rat kidney. Virchows Arch B Cell Pathol 22: 121-133.

[22] KRIZBAI IA ET AL. 2005. Blood-brain barrier changes during compensated and decompensated hemorrhagic shock. Shock 24: 428-433.

[23] LEI Y, PENG X, LIU L, DONG Z & LI T. 2015. Beneficial effect of cyclosporine A on traumatic hemorrhagic shock. J Surg Res 195: 529-540.

[24] LI L, LING Y, HUANG M, YIN T, GOU S, ZHAN N, XIONG J, WU H, YANG Z & WANG C. 2015. Heparin inhibits the inflammatory response induced by LPS and HMGB1 by blocking the binding of HMGB1 to the surface of macrophages. Cytokine 72: 36-42.

[25] LIBERT N, HARROIS A & DURANTEAU J. 2016. Haemodynamic coherence in haemorrhagic shock. Best Pract Res Clin Anaesthesiol 30: 429-435.

[26] LIN B, GAO Y, LI Z, ZHANG Z, LIN X & GAO J. 2020. Cannabidiol alleviates hemorrhagic shock-induced neural apoptosis in rats by inducing autophagy through activation of the PI3K/AKT pathway. Fundam Clin Pharmacol 34: 640-649.

[27] LIU F-C, LIU F-W & YU H-P. 2011. Ondansetron attenuates hepatic injury via p38 MAPK-dependent pathway in a rat haemorrhagic shock model. Resuscitation 82: 335-340.

[28] MANN DV, ROBINSON MK, ROUNDS JD, DEROSA E, NILES DA, INGWALL JS, WILMORE DW & JACOBS DO. 1997. Superiority of blood over saline resuscitation from hemorrhagic shock: a 31P magnetic resonance spectroscopy study. Ann Surg 226: 653-661.

[29] MARTÍNEZ RJÁ ET AL. 2013. Evolution and complications of chest trauma. Arch Bronconeumol 49: 177-180.

[30] MINEI JP ET AL. 2012. The changing pattern and implications of multiple organ failure after blunt injury with hemorrhagic shock. Crit Care Med 40: 1129-1135.

[31] MORRISON JJ, STANNARD A, RASMUSSEN TE, JANSEN JO, TAI NRM & MIDWINTER MJ. 2013. Injury pattern and mortality of noncompressible torso hemorrhage in UK combat casualties. J Trauma Acute Care Surg 75: S263-S268.

[32] OLIVEIRA FRMB, ASSREUY J & SORDI R. 2022. The role of nitric oxide in sepsis-associated kidney injury. Biosci Rep 42: BSR20220093.

[33] OLIVEIRA FRMB, SOUSA SOARES E, PILLMANN RAMOS H, LÄTTIG-TÜNNEMANN G, HARMS C, CIMAROSTI H & SORDI R. 2024. Renal protection after hemorrhagic shock in rats: possible involvement of SUMOylation. Biochem Pharmacol 227: 116425.

[34] PATEL NM, OLIVEIRA FRMB, RAMOS HP, AIMARETTI E, ALVES GF, COLDEWEY SM, COLLINO M, SORDI R & THIEMERMANN C. 2023. Inhibition of Bruton’s tyrosine kinase activity attenuates hemorrhagic shock-induced multiple organ dysfunction in rats. Ann Surg 277: e624-e633.

[35] PATEL NM ET AL. 2022. Inhibition of macrophage migration inhibitory factor activity attenuates haemorrhagic shock-induced multiple organ dysfunction in rats. Front Immunol 13: 886421.

[36] POTJE SR ET AL. 2021. Heparin prevents in vitro glycocalyx shedding induced by plasma from COVID-19 patients. Life Sci 276: 119376.

[37] PROBST C, PAPE H-C, HILDEBRAND F, REGEL G, MAHLKE L, GIANNOUDIS P, KRETTEK C & GROTZ MRW. 2009. 30 years of polytrauma care: an analysis of the change in strategies and results of 4849 cases treated at a single institution. Injury 40: 77-83.

[38] PROCTOR JL, SCUTELLA D, PAN Y, VAUGHAN J, ROSENTHAL RE, PUCHE A & FISKUM G. 2015. Hyperoxic resuscitation improves survival but worsens neurologic outcome in a rat polytrauma model of traumatic brain injury plus hemorrhagic shock. J Trauma Acute Care Surg 79: S101-S109.

[39] SORDI R, BOJKO L, OLIVEIRA FRMB, ROSALES TO, SOUZA CF, MORENO LW, ALVES GF, CARLOS J, VELLOSA R, FERNANDES D & GOMES JR. 2024. Doxycycline reduces liver and kidney injuries in a rat hemorrhagic shock model. Intensive Care Med Exp 12: 2.

[40] SORDI R, CHIAZZA F, COLLINO M, ASSREUY J & THIEMERMANN C. 2016. Neuronal nitric oxide synthase is involved in vascular hyporeactivity and multiple organ dysfunction associated with hemorrhagic shock. Shock 45: 525-533.

[41] SORDI R, CHIAZZA F, PATEL NSA, DOYLE RA, COLLINO M & THIEMERMANN C. 2015. ‘Preconditioning’ with low dose lipopolysaccharide aggravates the organ injury/dysfunction caused by hemorrhagic shock in rats. PLoS ONE 10: e0122096.

[42] SORDI R, NANDRA KK, CHIAZZA F, JOHNSON FL, CABRERA CP, TORRANCE HD, YAMADA N, PATEL NSA, BARNES MR, BROHI K, COLLINO M & THIEMERMANN C. 2017. Artesunate protects against the organ injury and dysfunction induced by severe hemorrhage and resuscitation. Ann Surg 265: 408-417.

[43] STARK K. 2021. Interplay between inflammation and thrombosis in cardiovascular pathology. Nat Rev Cardiol 18: 666-682.

[44] SWANK GM & DEITCH EA. 1996. Role of the gut in multiple organ failure: bacterial translocation and permeability changes. World J Surg 20: 411-417.

[45] THAKKAR B & WU GY. 2025. Ketamine hepatotoxicity: an underappreciated cause of liver damage - analysis by RUCAM. J Clin Transl Hepatol 13: 524-531.

[46] THIEMERMANN C, SZABO C, MITCHELL JA & VANE JR. 1993. Vascular hyporeactivity to vasoconstrictor agents and hemodynamic decompensation in hemorrhagic shock is mediated by nitric oxide. Proc Natl Acad Sci USA 90: 267-271.

[47] THOMPSON JS, BROWN SA, KHURDAYAN V, ZEYNALZADEDAN A, SULLIVAN PG & SCHEFF SW. 2002. Early effects of tribromoethanol, ketamine/xylazine, pentobarbital, and isoflurane anesthesia on hepatic and lymphoid tissue in ICR mice. Comp Med 52: 63-67.

[48] TREMOLEDA JL, WATTS SA, REYNOLDS PS, THIEMERMANN C & BROHI K. 2017. Modeling acute traumatic hemorrhagic shock injury: challenges and guidelines for preclinical studies. Shock 48: 610-623.

[49] TRENTZSCH H, NIENABER U, BEHNKE M, LEFERING R & PILTZ S. 2014. Female sex protects from organ failure and sepsis after major trauma haemorrhage. Injury 45: S20-S28.

[50] TYRRELL DJ, HORNE AP, HOLME KR, PREUSS JMH & PAGE CP. 1999. Heparin in inflammation: potential therapeutic applications beyond anticoagulation. Adv Pharmacol 46: 151-208.

[51] VANZANT EL ET AL. 2015. Advanced age is associated with worsened outcomes and a unique genomic response in severely injured patients with hemorrhagic shock. Crit Care 19: 77.

[52] VEILLEUX-LEMIEUX D, CASTEL A, CARRIER D, BEAUDRY F & VACHON P. 2013. Pharmacokinetics of ketamine and xylazine in young and old Sprague-Dawley rats. J Am Assoc Lab Anim Sci 52: 567-570.

[53] WRBA L, PALMER A, BRAUN CK & HUBER-LANG M. 2017. Evaluation of gut-blood barrier dysfunction in various models of trauma, hemorrhagic shock, and burn injury. J Trauma Acute Care Surg 83: 944-953.

[54] WU R ET AL. 2008. Human vasoactive hormone adrenomedullin and its binding protein rescue experimental animals from shock. Peptides 29: 1223-1230.

[55] ZHANG LM, ZHANG DX, FU L, LI Y, WANG XP, QI MM, LI CC, SONG PP, WANG XD & KONG XJ. 2019. Carbon monoxide-releasing molecule-3 protects against cortical pyroptosis induced by hemorrhagic shock and resuscitation via mitochondrial regulation. Free Radic Biol Med 141: 299-309.