The regulatory effects of serum catecholamines and endothelial cells in pig hemorrhagic shock and fluid resuscitation models

Abstract

Background

Acute blood loss not only leads to systemic compensatory response, but also the induced changes in vascular endothelial function.These pathological changes may have potential compensatory significance for maintaining organ perfusion and fluid resuscitation.

Objective

To understand trauma-induced endotheliopathy and their compensatory roles in acute hemorrhage, a porcine model of hemorrhagic shock (HS) was used to evaluate changes in vascular endothelial factors and catecholamine levels at different time points from shock to fluid resuscitation. Methods: HS was induced in female pigs by rapid bleeding via the arterial sheath. Hemodynamic monitoring was performed using a pulse index continuous cardiac output (PiCCO) system in HS and fluid resuscitation. Femoral vein blood samples were collected at baseline and 40% mean arterial pressure (MAP, shock), MAP recovery, and 30 min, 1 h, and 2 h after recovery. Serum levels of catecholamine and Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), Tie-2, Eselectin, intracellular adhesion molecule-1 (ICAM-1), soluble thrombomodulin (sTM), and Syndecan-1 (SDC-1) were evaluated using enzyme-linked immunosorbent assay (ELISA).

Results

Serum catecholamine levels were significantly higher in the shock than in the baseline state. Ang-1 and Ang-2 are endothelial growth factors secreted with distinct roles. Ang-1 stabilizes the endothelium and inhibits vascular leakage, and Ang-2 has the opposite effect. The ratio of Ang-2/Ang-1 was significantly higher in the shock state than in the baseline state; however, the Ang-1/Tie-2 ratio was comparable between the two states. This suggests that changes in vascular permeability may mainly depend on the upregulation of Ang-2 function. Serum levels of E-selectin, ICAM-1, sTM, and SDC-1 were significantly higher in the shock state than in the baseline state. After the MAP was restored to the baseline state, the levels of E-selectin, and SDC-1 remained higher compared with the baseline state until 1 h after MAP recovery. Conclusions: serum levels of catecholamines and vascular endothelial markers increased transiently under HS, promoting a compensatory response of the circulatory system to acute bleeding. This may be one of the potential theoretical basis for restrictive fluid resuscitation.

Introduction

Hemorrhagic shock (HS) is a type of hypovolemic shock with multiple causes, including trauma, maternal bleeding, gastrointestinal bleeding, perioperative bleeding, and arterial aneurysm rupture.1, 2 A rapid decrease in blood volume often triggers a series of compensatory responses in the body, such as an increase in heart rate (HR) and redistribution of blood flow, to maintain blood supply to vital organs.³ The systemic compensatory response to acute blood loss, such as sympathetic nervous system excitation, has been fully recognized. However, at the microcirculation level, the existence of other compensatory mechanisms besides blood flow redistribution caused by capillary contraction remains uncertain.

The vascular endothelium plays a crucial role in maintaining organ homeostasis by regulating vascular tone, coagulation, inflammation, and barrier function,⁴ and its function may change in various disease states. In sepsis, a large amount of inflammatory mediators may lead to endothelial damage, tissue hypoperfusion, disseminated intravascular coagulation, and organ dysfunction. After trauma, animal models show increased pulmonary vascular permeability following hemorrhagic shock⁵ or surrogate markers of endothelial dysfunction, such as glycocalyx shedding.⁶ Therefore, the dysregulation of normal endothelial function is a significant pathological characteristic of organ dysfunction during disease states. Serum levels of vascular endothelial biomarkers can accurately reflect endothelial function and injury in real time. Their fluctuations have been confirmed to be related to various conditions, including sepsis,⁷ chronic kidney disease,⁸ peripheral artery disease,⁹ and heart failure.¹⁰

To understand trauma-induced endotheliopathy and their roles in acute hemorrhage, in this study, we utilized a porcine model of hemorrhagic shock (HS) to examine the variations in serum levels of vascular endothelial factors and catecholamines at different time points, spanning from the onset of shock to fluid resuscitation, and attempt to explore the significance of endothelial function adjustment in acute blood loss and its impact on subsequent fluid resuscitation.

Materials and methods

Animal preparation

Ten healthy domestic female Beijing Landrace pigs (67.4 ± 1.1 days old, weighing 34.4 ± 1.0 kg) were provided by a registered laboratory animal center in Beijing, China. This study was conducted in strict accordance with the animal care and application guidelines established by the Animal Care and Use Committee (No. 2022–04-054). Animal experiments conformed to the Guidelines for the Care and Use of Animals as expressed in the Declaration of Helsinki.¹¹ The animals were fasted overnight with free access to water and initially sedated using intramuscular injection of midazolam (2 mg/kg). Anesthesia was maintained with continuous intravenous infusion of propofol (2–3 mg/kg/hr). A cuffed 6.5 mm endotracheal tube was advanced into the trachea. Animals were mechanically ventilated using a volume-controlled ventilator (Mindray SV300, Shenzhen, China), with a tidal volume of 10 ml/kg and a fraction of inspired oxygen (FiO₂) of 0.21. The left femoral artery was dissected and a 4-F arterial catheter was inserted into the descending aorta to measure the mean arterial pressure (MAP). A 6-F arterial sheath was inserted in the right femoral artery for rapid bleeding. A 5-F central venous catheter was inserted through the left femoral vein for volume resuscitation. The room temperature was adjusted to 26 °C. The body temperature was maintained above 37 °C with a heating pad.

Experimental protocol

After the operation, the animals were allowed to stabilize for 30 min, and baseline data were obtained. HS was induced by rapid bleeding via the arterial sheath. HS was achieved if the cardiac index (CI) and MAP dropped below 40% of the baseline value.¹² Shed blood was collected in sterile bags (S-400, Sichuang Nightingale Biological Co., Ltd., China). Fluid resuscitation was started after 30 min of shock. The animals were resuscitated with the shed blood and then received a basal crystalloid infusion of 30 ml/kg/h. Fluid resuscitation was continued until MAP and CI returned to baseline values and maintained a stable state for 2 h. No vasoconstrictor drugs were used throughout the experimental process.

Measurements

Pulse index continuous cardiac output (PiCCO) hemodynamic parameters

Arterial and central venous catheters were connected to an integrated bedside monitor (PiCCO; Pulsion Medical Systems, Munich, Germany) for continuous hemodynamic monitoring. Hemodynamic parameters, including MAP, HR, stroke volume (SV), cardiac output (CO), central venous pressure (CVP), and extravascular lung water (EVLW) were measured at baseline and 40% MAP (shock), MAP recovery, and 30 min, 1 h, and 2 h after recovery (Fig. 1). The shock index (SI) was derived from the following formula: SI = heart rate/systolic pressure.

Fig. 1.

Experimental process and the time points of blood sample collection.

Serum levels of vascular endothelial injury biomarkers and catecholamines

Femoral vein blood samples were collected at baseline and shock, MAP recovery, and 30 min, 1 h, and 2 h after recovery. Serum levels of catecholamines and Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), Tie-2, E-selectin, intracellular adhesion molecule-1 (ICAM-1), soluble thrombomodulin (sTM), and Syndecan-1 (SDC-1) were evaluated using enzyme-linked immunosorbent assay (Beijing Function Biotechnology Co., Ltd, Beijing, China).

Statistical analysis

All statistical analyses were performed using SPSS 24.0 software (SPSS, Chicago, IL, USA). Continuous variables were expressed as mean ± standard deviation. One-way ANOVA followed by Post hoc (LSD) test was used to compare the differences in variables at different time points after hemorrhage to resuscitation. The normality of the distribution and equality of variance for continuous variables were assessed using the Kolmogorov-Smirnov test and the homogeneity of variance test, respectively. A P-value of less than 0.05 was considered statistically significant.

Results

Baseline characteristics and amounts and rates of hemorrhage of animals

The average weight and age of ten pigs were 34.4 ± 1.0 kg and 67.4 ± 1.1 days, respectively. The amount and rate of blood loss were 1019.8 ± 303.2 ml and 31.2 ± 12.8 minutes, respectively. The volume of blood transfused and the time for resuscitation (until MAP was restored to its baseline value) were 533.70 ± 228.97 ml and 53.8 ± 15.2 min, respectively. The amount of bleeding and resuscitation time for each animal is detailed in Table 1.

Table 1.

The volume of blood and resuscitate time of each animal.

Animal No	Weight（kg）	Age（d）	Blood volume removed (ml)	Blood transfusion volume (ml)	Crystalloid volume (ml)
1	33	68	762	368	165
2	35	68	760	255	170
3	33	68	1538	862	150
4	35	68	1146	500	175
5	35	69	1419	758	150
6	33	67	1049	677	145
7	35	67	712	389	150
8	35	65	1073	754	160
9	35	67	1100	729	113
10	35	67	639	245	160

Comparison of PiCCO parameters at different time points during hemorrhage and resuscitation

Upon reaching the target MAP, the animal suffered a blood loss equivalent to approximately 40% of its total blood volume. Following fluid resuscitation and blood transfusion, the body's blood volume was restored to approximately 84% of its initial baseline level. HR and SI were higher in the shock state than in the basal state (HR: 156.0 ± 44.5 vs. 115.1 ± 27.6 bpm, P = 0.019; SI: 2.4 ± 1.0 vs. 0.9 ± 0.3, P < 0.001), while SV and CO were significantly lower in the shock state than in the basal state (SV: 14.1 ± 7.2 vs. 36.0 ± 6.5 ml, P < 0.001; CO: 2.0 ± 0.6 vs. 4.4 ± 1.0 L/min, P < 0.001). Meanwhile, no difference in HR and SI was found after MAP recovery compared with baseline (HR: 133.0 ± 33.1 vs.115.1 ± 27.6 bpm, P = 0.132; SI: 1.2 ± 0.2 vs. 0.9 ± 0.3, P = 0.152), while SV and CO were significantly lower after MAP recovery compared with the basal state (SV: 24.2 ± 8.0 vs. 36.0 ± 6.5 ml, P = 0.021; CO: 3.3 ± 1.0 vs. 4.4 ± 1.0 L/min, P = 0.012). No differences in CVP and ELVW were found between the baseline and shock or fluid resuscitation (Fig. 2A–F).

Fig. 2.

Comparison of PiCCO parameters at different time points during hemorrhage and resuscitation. A) Heart rate (HR); B) Stroke volume (SV); C) Cardiac output(CO); D) Shock index (SI); E) Central venous pressure, (CVP); F) Extravascular lung water (EVLW). HR and SI were higher in the shock state than in the basal state (P_HR = 0.019; P_SI < 0.001), while SV and CO were lower in the shock state than in the basal state (P_SV < 0.001; P_CO < 0.001). After MAP recovery SV and CO were lower than the basal state (P_SV = 0.021; P_CO = 0.012). Compared with baseline, *P < 0.05; **P < 0.001.

Changes in serum levels of catecholamines

Serum levels of catecholamines were significantly higher in the shock state than in the basal state (380.4 ± 56.9 vs. 308.4 ± 62.8 ng/ml, P = 0.041). No significant difference in catecholamines concentration was detected between the two states after fluid resuscitation (Fig. 3).

Fig. 3.

Changes in serum levels of catecholamine during hemorrhage and resuscitation. Serum levels of catecholamine were significantly higher in the shock state than in the basal state (P = 0.041). Compared with baseline, *P < 0.05.

Changes in vascular endothelial injury biomarkers

Under shock state, the Ang-2/Ang-1 ratio and serum levels of E-selectin, ICAM-1, sTM, and SDC-1was higher in the shock state compared to the baseline state (the Ang-2/Ang-1 ratio: 7.5 ± 1.7 vs. 5.4 ± 1.1, P < 0.001; E-selectin: 37.9 ± 4.1 vs. 28.9 ± 2.5 ng/ml, P < 0.001; ICAM-1: 93.4 ± 7.6 vs. 81.6 ± 14.8 ng/ml, P = 0.014; sTM: 7.6 ± 1.1 vs. 6.2 ± 0.7 ng/ml, P = 0.001; SDC-1: 21.0 ± 1.6 vs. 14.8 ± 1.2 ng/ml, P < 0.001). However, there was no statistically significant difference in the Ang-1/Tie-2 ratio between the two states (4.6 ± 0.5 vs. 4.4 ± 1.0, P = 0.548).

From the MAP was restored to 1 hour later, serum levels of E-selectin (32.5 ± 3.4 vs. 28.9 ± 2.5 ng/ml, P = 0.036) and SDC-1 (17.7 ± 2.1 vs. 14.8 ± 1.2 ng/ml, P = 0.001) remained elevated compared to the baseline state (Fig. 4A–F).

Fig. 4.

Changes in vascular endothelial injury biomarkers during hemorrhage and resuscitation. A) The ratio of Angiopoietin-2 (Ang-2)/Angiopoietin-1 (Ang-1);B) The ratio of Ang-1/Tie-2; C) E-selectin;D) The intracellular adhesion molecule-1 (ICAM-1);E)The soluble thrombomodulin (sTM); F) Syndecan-1 (SDC-1). The ratio of Ang-2/Ang-1 was higher in the shock state than the baseline state (P < 0.001); Serum levels of E-selectin, ICAM-1, sTM, and SDC-1 were higher in the shock state than in the baseline state (P_E-selectin < 0.001; P _ICAM-1 = 0.014;P _sTM = 0.001; P_SDC-1 < 0.001).After the MAP was restored to the baseline state, serum levels of E-selectin (P = 0.036) and SDC-1 (P = 0.001) remained higher compared with the baseline state until 1 h after MAP recovery. Compared with baseline, *P < 0.05; **P < 0.001.

Discussion

The current study indicated that serum levels of catecholamines and endothelial function biomarkers increased dramatically during HS. The regulation of neuroendocrine and vascular endothelial functions may, to some extent, promote the compensatory effect of the circulatory system on hemorrhagic stress. These findings may provide a new theoretical basis for restrictive fluid resuscitation.

PiCCO was utilized for hemodynamic monitoring in the current study to assess whether and when the animal model met the shock criteria. During hemorrhage, the decrease in preload led to a significant reduction in SV and CO, and an increase in HR reactivity. During fluid resuscitation, when MAP returned to the baseline level, the transfusion volume of the shed blood and crystalloid was approximately 60% of the blood loss volume. This phenomenon is consistent with the previous description of the immediate and intermediate phases of hemorrhagic shock.³ The immediate stage usually triggers compensatory effects by stimulating the sympathetic nervous system, while the intermediate phase is characterized by transcapillary fluid shifts pulling fluids from interstitial and intracellular compartments into the vasculature.¹³ Due to these compensatory effects, even inadequate fluid or blood supplementation is sufficient to maintain perfusion of vital organs during fluid resuscitation.

Our study suggests that self-compensation may be associated with changes in endogenous catecholamines and the adjustment of endothelial function in hemorrhagic shock. Serum catecholamine levels significantly increase during the shock state. The reason behind this is that acute hemorrhage leads to a decrease in blood pressure, which stimulates the sympathetic nervous system by activating the baroreceptors and releasing endogenous catecholamines. Sudden increases in endogenous catecholamines can elevate heart rate, enhance myocardial contractility, and selectively constrict blood vessels, thereby improving MAP to a certain extent.¹³ However, this compensatory effect is limited and only occurs in the immediate phase of hemorrhagic shock.

The comparison of vascular endothelial biomarkers revealed that the adjustment of endothelial function may be a potential mechanism for self-compensation in the intermediate phase of hemorrhagic shock. Ang-1 and Ang-2 are endothelial growth factors secreted with distinct roles in regulating vascular quiescence.14, 15 Ang-1 stabilizes the endothelium and inhibits vascular leakage by constitutively activating the Tie-2 receptor. In contrast, Ang-2 disrupts microvascular integrity by blocking the Tie-2 receptor, leading to vascular leakage, which is a major mechanism of organ damage.14, 15 Our results showed that the Ang-2/Ang-1 ratio was significantly higher in the shock state than in the basal state. This indicates that the vascular endothelium tends to increase permeability during massive hemorrhage, allowing fluid to penetrate the blood vessels as a form of self-supplementation in the tissue gaps. Since there was no significant change in the Ang-1/Tie-2 ratio before and after blood loss and during fluid resuscitation, we speculate that the alteration in vascular permeability may primarily depend on the upregulation of Ang-2 function.

E-selectin is a glycoprotein adhesion molecule that is specifically expressed on activated endothelial cells. It mediates the adhesion and rolling of leukocytes on the endothelium.16, 17 ICAM-1 is widely distributed on the surface of hematopoietic and peripheral hematopoietic cells, and regulates barrier function and modulates endothelial permeability by controlling cytokine production.¹⁸ Both biomarkers reflect the pathological process of endothelial cell activation. Under physiological conditions, endothelial cells play a crucial role in regulating coagulation and perfusion through their essential role in vasomotor control.¹⁹ Meanwhile, they form a barrier that plays a central role in controlling permeability and regulating the distribution of water, cells, and molecules from the circulation into tissues. Our results confirm that both E-selectin and ICAM-1 levels in hemorrhagic shock are significantly elevated compared to baseline. This indicates that acute hemorrhage may cause changes in endothelial function, leading to a series of stress reactions such as increased permeability and promotion of thrombosis formation in response to traumatic stimuli.

Positioned at the interface between blood and the endothelial surface, the glycocalyx is central to maintaining a healthy endothelium. The endothelial glycocalyx serves as a physical barrier to circulating platelets, leukocytes, and soluble endothelial cell activating factors, protecting the underlying endothelium. SDC-1 and sTM are the major components of the endothelial glycocalyx, which is a layer of membrane-bound macromolecules on the luminal surface of the vascular endothelium.²⁰ In cases of major injury and hemorrhagic shock, syndecans are enzymatically cleaved from the cell surface by sheddases, leading to a noticeable thinning of the entire glycocalyx layer.21, 22 Therefore, an increase in serum SDC-1 and sTM levels indicates damage to the endothelial surface glycocalyx and a weakening of the endothelial barrier.23, 24, 25 This structural change is a manifestation of endothelial damage during hemorrhagic shock, and it is also the pathological basis for alterations in endothelial function, which are essential for maintaining proper hemostasis, promoting coagulation at the cell surface, compromising vascular barrier integrity, and facilitating leukocyte adhesion and transmigration.

Our results showed that the levels of endogenous catecholamines and serum endothelial biomarkers both returned to baseline levels after fluid resuscitation. This indicates that these changes are not only pathological damage caused by acute hemorrhage but also the physiological basis for the circulatory system to self-compensate. Whether it is the excitement of the sympathetic nervous system in the immediate phase or the regulation of endothelial function in the intermediate phase,²⁶ both contribute to the utilization of limited fluid to maintain perfusion of key organs. This may be one of the potential theoretical bases for restrictive fluid resuscitation.

However, this study has several limitations. Firstly, the small sample size may lead to results that deviate from those of a single female animal, and there may be differences in the degree of stress response between adult and young pigs. Therefore, further observational studies are needed to describe the pathological process. Furthermore, additional controlled trials should be conducted to establish a theoretical basis for treatment. Secondly, procedures such as surgery and catheterization may stimulate animals, impacting the levels of catecholamines and endothelial biomarkers. Therefore, future studies should strictly utilize minimally invasive procedures to overcome this challenge.

Conclusion

In summary, the current study found that serum levels of catecholamines and vascular endothelial biomarkers transiently increased in hemorrhagic shock, prompting a compensatory response from the circulatory system to acute bleeding. This may be one of the potential theoretical basis for restrictive fluid resuscitation.

Financial support

This study is supported by the National Natural Science Foundation of China (Grant No. 82274131).

Ethics approval and consent to participate

This study was carried out in strict adherence to the animal care and application guidelines established by the Animal Care and Use Committee of Capital Medical University (No.2022-04-054). Animal experiments followed the Guidelines for the Care and Use of Animals as articulated in the Declaration of Helsinki.

CRediT authorship contribution statement

Xiaoli Zhao: Methodology, Investigation, Data curation. Wei Yuan: Writing – original draft, Investigation, Formal analysis, Data curation. Shuo Wang: Formal analysis, Data curation. Junyuan Wu: Methodology, Investigation, Data curation. Chunsheng Li: Writing – review & editing, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.