EXPERIMENTAL RAT MODELS OF HEMORRHAGIC SHOCK: INSIGHTS INTO CONTROLLED, UNCONTROLLED, AND TRAUMA-ASSOCIATED PROTOCOLS

Hemorrhagic shock (HS) is a severe form of hypovolemic shock caused by progressive blood loss, in which failure of compensatory autonomic and cardiovascular mechanisms leads to circulatory collapse and impaired tissue perfusion (1). Although hemorrhagic shock may arise from a variety of causes, traumatic injury remains the most prevalent etiology. Traumatic hemorrhage is a major contributor to mortality in both civilian and military populations, accounting for approximately one-third of all trauma-related deaths (2).

Much of the current understanding of HS stems from decades of research using animal models (Fig. 1). These experimental systems have been indispensable in elucidating the complex pathophysiological processes underlying shock, including tissue hypoperfusion, systemic inflammation, coagulopathy, and multiorgan failure (3, 4). Moreover, animal models have provided a vital platform for the development and preclinical evaluation of novel therapeutic strategies, such as advanced resuscitation protocols and pharmacologic interventions (5).

Fig. 1. Experimental models of hemorrhagic shock (HS).

When selecting an appropriate model for experimental hemorrhagic shock, researchers must carefully weigh both its advantages and limitations (6). The ideal model should be straightforward to implement, ethically and logistically feasible, and capable of accurately replicating the clinical features of human hemorrhagic shock. Furthermore, it should demonstrate high levels of reproducibility and standardization to allow meaningful comparisons across studies (7).

Beyond summarizing the available models, a central objective of this review was to provide a comparative analysis of their advantages, limitations, and translational relevance, thereby assisting investigators in selecting the most appropriate model aligned with their specific research questions and experimental aims (Table 1).

Table 1. Selection guide for rodent hemorrhagic shock models based on experimental outcome.

Legend: model is most appropriate for this objective; model is appropriate for this objective; model is suboptimal or not recommended. Abbreviations: HS, hemorrhagic shock; UHS, uncontrolled hemorrhagic shock; T/HS, traumatic hemorrhagic shock; TBI, traumatic brain injury; CNS, central nervous system; MAP, mean arterial pressure.

METHODS

To gather relevant literature, a targeted search of the electronic databases PubMed, Google Scholar, Embase, and ResearchGate was performed. The search focused on articles published in peer-reviewed journals, primarily within the last two decades, although seminal older studies were included when deemed relevant to experimental design or historical context. The literature search was conducted with the aim of identifying both foundational and recent studies that provide detailed descriptions of hemorrhage induction methods, trauma combinations, and resuscitation strategies. The following keywords and combinations thereof were used to identify pertinent studies: “Hemorrhagic shock”, “Rat models”, “Controlled hemorrhage”, “Uncontrolled hemorrhage”, “Trauma models”, “Traumatic brain injury”, “Resuscitation”, “Experimental design”, and “Hemorrhagic shock combined with trauma”. Boolean operators (AND, OR) were applied to refine and structure the searches appropriately. The final search was completed on June 15, 2025. Studies were selected based on their relevance to experimental rat models of HS. Inclusion criteria required a clear description of the hemorrhage induction method, trauma mechanisms (if applicable), resuscitation approach (if applicable), and measurable physiological or biochemical outcomes. Preference was given to studies with well-defined protocols and appropriate anesthesia and monitoring techniques. Studies were excluded if they contained an incomplete description of the HS model, provided a vague or unclear definition of the animal species, used species other than rats (e.g., large animals, mice, or non-rodent models), or lacked sufficient methodological detail. References were additionally identified through manual screening of bibliographies of included papers to ensure comprehensive coverage of the topic. Given the narrative nature of this review, no formal quality assessment or risk of bias analysis was performed; instead, emphasis was placed on diversity of experimental approaches, model reproducibility, and translational relevance.

MODEL PARAMETERS AND EXPERIMENTAL VARIABLES

Strain selection - inbred or outbred?

Among the key considerations in model selection is the choice of animal species and strain, as both genetic background and physiological characteristics can significantly influence the response to hemorrhage and therapeutic interventions. Rats (Rattus norvegicus) are widely used in experimental research, including HS studies, due to their manageable size, ease of handling, and, more importantly, their physiological and genetic similarities to humans (8, 9). Notably, certain immune responses to hemorrhage in rats have been reported to parallel those observed in humans (10). However, scientists need to be aware of the limitations of rat models. Key interspecies differences-particularly in the coagulation cascade (6) and cardiovascular responses-can affect the extrapolation of findings to human physiology (11). These disparities necessitate careful interpretation of data and underscore the importance of selecting appropriate strains and experimental conditions when modeling HS in rats.

In the context of scientific research, the selection of rat strains is based on either inbreeding or outbreeding. Inbred strains are genetically homogeneous, with individuals sharing almost identical genetic material apart from a limited number of spontaneous mutations that may have accumulated over time. In contrast, outbred rats exhibit greater genetic variability, which more closely reflects the heterogeneity of human populations and may enhance the external validity of experimental findings (12).

A number of dedicated registries have been established to document rat strains and their characteristics (13). In experimental research on hemorrhagic shock, the Sprague-Dawley and Wistar strains are most frequently employed, owing to their docile temperament, broad availability, and well-characterized physiological profiles (14, 15). Importantly, both strains are outbred, a feature that introduces substantial genetic variability. This variability can be advantageous, as it better reflects the heterogeneity of responses observed in human populations. Nonetheless, subtle physiological differences exist between the two. For example, Wistar rats may exhibit distinct baseline cardiovascular or inflammatory parameters compared with other strains, potentially influencing experimental outcomes. By contrast, inbred strains such as Lewis or Fischer 344 are typically chosen when genetic uniformity is required, particularly in studies focused on specific immunological or molecular pathways. The selection of rat strain has been shown to significantly influence HS models, particularly in terms of blood pressure dynamics, inflammatory responses, and susceptibility to organ injury (6, 16).

Beyond commonly used laboratory strains, selectively bred lines have also been employed to investigate how specific physiological traits affect HS outcomes. Klemcke et al. examined high-capacity runners (HCR) and low-capacity runners (LCR), which were developed through divergent selection for aerobic exercise performance (17). While HCR rats were expected to demonstrate enhanced survival due to superior oxygen utilization, the study found no significant difference in survival times between the two groups. These findings indicate that aerobic capacity alone does not determine early outcomes in HS and highlight the contribution of additional mechanisms such as neuroendocrine responses and blood flow regulation. Although outbred strains are more commonly adopted, Festing et al. highlighted the advantages of inbred rats, emphasizing that their defined genetic background improves reproducibility and enables a clearer attribution of observed effects to the experimental intervention (18).

Translational considerations

Rat models have substantially advanced the understanding of shock physiology and remain indispensable for mechanistic and therapeutic research. Nevertheless, important interspecies and experimental differences must be acknowledged to interpret findings reliably and to translate them effectively into clinical practice.

Rats differ from humans in several physiological domains relevant to the clinical trajectory of shock and organ failure (6). They have a higher baseline heart rate and metabolic rate, while their compensatory responses to blood loss differ in magnitude and duration. This is exemplified by the absence of a heart rate increase with a decrease in mean arterial pressure (MAP) observed in various models of HS. (14, 19). However, this phenomenon may also be a consequence of isoflurane anesthesia, as studies employing a combination of ketamine and xylazine for anesthesia demonstrated an adequate increase in heart rate (20).

Differences in coagulation and hemostasis further affect translational validity. Rats vary from humans in coagulation factor composition, platelet count and function, as well as fibrinolytic activity, which can influence both the onset and resolution of coagulopathy and the apparent efficacy of hemostatic therapies (21). Immunological differences, including cytokine kinetics and leukocyte activation, can likewise alter the post-shock inflammatory trajectory (10). Rodents also demonstrate more pronounced metabolic responses to hemorrhage, with higher lactate and succinate concentrations than in humans (22).

Finally, most experiments use young, healthy, genetically homogenic, anesthetized animals exposed to controlled hemorrhage and immediate resuscitation under normothermic, aseptic conditions. These designs omit clinical variables such as advanced age, comorbidity, delayed intervention, and polytrauma that shape outcomes in human patients. Moreover, anesthetic and analgesic agents themselves modulate cardiovascular, coagulative, and inflammatory pathways, potentially confounding treatment effects. Recognition of these limitations does not diminish the scientific value of rat models but emphasizes the need for careful model selection, transparent reporting, and cautious extrapolation.

Anesthetic agents

Within the ethical framework of the 3Rs principle, refinement emphasizes the use of adequate anesthesia to minimize pain and distress. In rat HS models, both inhalational and injectable anesthetics are used. Inhalational agents such as isoflurane (most commonly) and sevoflurane offer rapid onset, easy control of anesthetic depth, and quick recovery. Importantly, they exert relatively mild cardiovascular depression compared with injectable agents, making them preferable in studies requiring hemodynamic stability (23-26). Injectable regimens such as ketamine-xylazine (27, 28) or sodium pentobarbital (29, 30) remain widely used, particularly in settings without access to inhalation systems. Their simplicity of administration is advantageous, but they are associated with more pronounced respiratory and cardiovascular depression. The anesthetic choice can significantly impact immune responses: inhalational agents such as isoflurane and sevoflurane induce smaller cytokine alterations compared with ketamine-xylazine, which may confound immunological outcomes (31, 32).

Newer agents, including alfaxalone, are increasingly adopted due to predictable pharmacodynamics and minimal cardiovascular effects, often in combination with sedatives or analgesics. By contrast, urethane is now largely restricted to non-survival neurophysiological studies because of its carcinogenicity despite providing stable anesthesia (33, 34). Historically, halothane was used in rats but has been abandoned owing to hepatotoxicity, cardiovascular depression, and arrhythmogenic potential (35-37). Thus, anesthetic selection should balance methodological requirements (e.g., hemodynamic stability, immunological readouts) with ethical obligations to ensure animal welfare (38).

Conscious (unanesthetized) models

When planning an experiment, it is important to consider that the use of anesthetics in animal models may interfere with the assessment of key hemodynamic responses and outcome parameters (39). To minimize these confounding effects, some investigators have adopted conscious animal models in which physiological responses to hemorrhage are studied in awake, non-anesthetized animals (40). While technically demanding, costly and accompanied by additional ethical considerations, such models enable the evaluation of integrated systemic and behavioral responses to blood loss, providing data that are more reflective of human pathophysiology. Conscious models are therefore particularly valuable for studying survival, hemodynamic adaptation, and resuscitation efficacy under conditions that closely simulate clinical scenarios of uncontrolled or prehospital hemorrhage (41).

Analgesic agents

Analgesia in rat HS models is equally critical, as pain management must balance welfare obligations with preservation of experimental endpoints such as hemodynamics, coagulation, and inflammation. Buprenorphine, a partial μ-opioid receptor agonist, is widely used for survival surgeries due to its long duration of action and limited cardiovascular effects (42). However, in HS settings it may reduce survival (43), alter clot strength (44), and induce hyperalgesia with repeated dosing (45).

Non-steroidal anti-inflammatory drugs (NSAIDs) are common alternatives. Meloxicam provides effective analgesia with minimal acute hemodynamic impact, though prolonged use carries risks of nephrotoxicity, gastrointestinal irritation, and coagulation changes. Carprofen is often favored in HS protocols because of its relative safety for cardiovascular and inflammatory endpoints (46).

Analgesic use depends on study design. In survival models, analgesia is mandatory under institutional and international guidelines (e.g., ARRIVE) (38). In non-survival models, where animals remain under deep anesthesia until euthanasia, systemic analgesics may be omitted to avoid confounding data. Ultimately, analgesic regimens must be tailored to balance humane treatment with experimental validity.

Vascular access and cannulation techniques

Proper vascular cannulation is a critical step in experimental procedures using hemorrhagic shock (HS) models. The selection of cannulation sites and the number of vascular access points should be tailored to the specific objectives of each study. Typically, catheters are inserted into either the jugular vein and carotid artery or the femoral vein and femoral artery. When additional physiological parameters, such as cardiac function, need to be monitored, cannulation of three vessels is generally required. Comprehensive protocols for jugular vein, carotid artery, and femoral artery cannulation have been outlined by Feng et al. (47) and Jespersen et al. (48).

Among these options, femoral artery cannulation is often preferred over carotid artery access, as the latter has been linked to a higher incidence of seizures and markedly poorer survival outcomes. The lower risk of neurological complications and improved survival associated with femoral artery access make it a safer and more reliable method for arterial pressure monitoring in HS models (49).

All catheterization procedures should be performed under general anesthesia, with continuous monitoring of key physiological parameters, including core body temperature and respiratory rate. Because anesthesia commonly induces hypothermia, the use of external warming devices, such as heating pads, is essential to maintain normothermia throughout the procedure.

Use of anticoagulants in hemorrhagic shock research

In the early studies on hemorrhagic shock, systemic anticoagulation with heparin was routinely employed, and for a considerable period it was the standard in experiments with controlled hemorrhage (50). However, this practice was called into question following the publication of research results indicating that systemic heparinization can significantly impact the progression of shock, both by protecting microcirculation and modulating organ response (51, 52). Rana and Wang demonstrated that systemic administration of heparin preserves microcirculation patency and may protect organs during HS. This was a significant argument that stimulated the development of alternative experimental protocols (53). Consequently, systemic heparinization is now reserved for selected models specifically investigating microcirculatory protection or coagulopathy mechanisms. In most contemporary studies, regional anticoagulation - limited to catheter flushing or citrate-treated autotransfused blood - is preferred, as it minimizes interference with endogenous hemostatic processes while maintaining experimental reliability and translational relevance (53, 54).

Duration of hemorrhagic shock

The duration of the shock phase in experimental models is deliberately varied to reflect the clinical realities of transporting patients after trauma. In major metropolitan areas with advanced emergency medical infrastructure, the time interval from injury to admission to the emergency department is typically approximately 30 minutes, and in certain circumstances, it can be even shorter (55, 56). However, in situations involving combat, mass disasters or rural areas, the time required to reach a facility capable of providing surgical treatment can be many hours (57, 58). Prolonged shock has been shown to significantly increase the risk of further decompensation and mortality. This is the basis for the concept of the “golden hour”- a term that emphasizes the need to control bleeding and implement causal treatment as quickly as possible (55, 59). Data from civil and military registries indicate that most deaths from uncontrolled hemorrhage occur within the first 30 minutes of injury, and that prolonging the time to surgical intervention or transfusion significantly worsens the prognosis (57). Clinical studies have shown that transport times exceeding 20 minutes are associated with higher mortality, especially when plasma or blood transfusions are not available in the pre-hospital phase (60). Consequently, both brief and protracted periods of shock are employed in animal models to investigate the consequences of delayed resuscitation, organ complications, and to evaluate novel therapeutic strategies that may extend the “survival window” in prolonged pre-hospital care settings (61).

Contemporary fluid resuscitation strategies in hemorrhagic shock

Modern fluid resuscitation strategies for HS are based on the principle of restricted fluid administration until definitive control of bleeding is achieved. The preferred fluids are balanced crystalloids (e.g., Ringer’s lactate), whereas colloids - particularly hydroxyethyl starch (HES) - are not recommended due to the increased risk of renal impairment and coagulopathy. As demonstrated in a number of studies, albumin provides no benefit in comparison to crystalloids (62, 63). Moreover, its utilization in patients with traumatic brain injury (TBI) may be deleterious, although this may be attributable to the fatty acids added to pharmaceutical albumin, rather than to the albumin itself (64). In cases of massive hemorrhage, the transfusion of blood components is recommended, especially within massive transfusion protocols. Both experimental and clinical data demonstrate that the transfusion of whole blood or blood components more effectively reduces endothelial injury and improves survival compared to crystalloids alone (1, 65). In selected scenarios, particularly in prehospital or military environments, small-volume hypertonic fluids and experimental formulations (e.g., ALM (Adenosine, Lidocaine, and Magnesium)) have been explored to achieve hemodynamic stabilization with minimal fluid volume (66). However, early prehospital vasoconstrictor administration (including vasopressin) is not recommended, as clinical data indicate an association with increased mortality (67). The concept of permissive hypotensive resuscitation, applied in both preclinical and translational studies, involves limiting fluid administration to maintain a systolic blood pressure of 70–80 mmHg (or mean arterial pressure of 50–60 mmHg) until hemorrhage is controlled. This approach aims to minimize dilutional coagulopathy, avoid disruption of forming clots, and reduce complications associated with excessive fluid administration (68, 69). Meta-analyses and guidelines from the American Association for the Surgery of Trauma (AAST) and the American College of Surgeons Committee on Trauma (ACS-COT) highlight improved survival, reduced blood product usage, and less blood loss with this strategy compared to traditional liberal fluid therapy. An important exception includes patients with TBI, in whom higher perfusion pressures should be maintained to ensure adequate cerebral blood flow (70). In both clinical and experimental settings, dynamic indicators of fluid responsiveness - such as stroke volume variation, capillary refill time, and lactate concentration - are increasingly recognized as essential parameters for guiding individualized, goal-directed fluid resuscitation and optimizing hemodynamic management (63).

The timing of resuscitation varies across studies, with early initiation (immediately after target MAP or volume loss) favoring hemodynamic recovery but potentially obscuring the natural compensatory phase, whereas delayed resuscitation, initiated 30–90 min after hemorrhage, is commonly employed to simulate prehospital delays and evaluate prolonged hypoperfusion tolerance (71).

The infusion rate is also critical. Rapid bolus administration transiently restores perfusion but is associated with increased risk of endothelial injury, dilutional coagulopathy, and rebound hypotension. Slow, titrated infusions maintain more stable pressures and reduce total fluid requirements, minimizing complications such as fluid overload and coagulopathy (72).

CLASSIFICATION AND EXPERIMENTAL UTILITY
OF HEMORRHAGIC SHOCK MODELS

HS models are generally categorized according to the method of hemorrhage induction, with controlled and uncontrolled paradigms offering distinct advantages depending on the research context. Controlled HS models are widely used in scientific research and are typically classified as either fixed-pressure or fixed-volume models. These approaches are particularly valuable for investigating the pathophysiology of HS and for evaluating the efficacy of novel therapeutic interventions under standardized and reproducible conditions.

In contrast, uncontrolled HS models aim to more closely mimic the spontaneous blood loss observed in real-world trauma, capturing the dynamic and unpredictable nature of hemorrhage resulting from vascular injuries. In studies combining traumatic brain injury (TBI) with HS, researchers may select either a controlled or uncontrolled model depending on the study objectives, such as mechanistic exploration, pharmacological testing, or translational relevance.

Controlled hemorrhagic shock models

Controlled hemorrhage models are defined by the precise regulation of shock-inducing parameters, including arterial blood pressure, total blood volume, or the rate of blood withdrawal. These models enable real-time monitoring of essential physiological parameters such as heart rate, body temperature, and respiratory rate, facilitating detailed analysis of the systemic effects of hemorrhage under standardized conditions.

Fixed-pressure hemorrhagic shock

The fixed-pressure model is one of the most widely used approaches for studying controlled hemorrhage. Its foundational principles were first established by Penfield (73), and later, in 1942, Wiggers refined these concepts by developing the classic fixed-pressure hemorrhage model, which has since served as the basis for numerous modifications in experimental research (50). In this model, blood is withdrawn until a predetermined MAP is reached. Once the target pressure is achieved, it is maintained over a specified period, either through additional blood withdrawal or via reinfusion of shed blood or resuscitation fluids, depending on the experimental design. Continuous blood pressure monitoring allows for precise control of shock severity throughout the procedure. Experimental data suggest that a slower rate of initial hemorrhage may increase survival by allowing partial physiological compensation. However, this approach may reduce the clinical relevance of the model, as real-world traumatic hemorrhage is typically rapid and severe.

Currently, there is no consensus regarding the optimal MAP or shock duration required to establish a reproducible hemorrhagic shock model. Nonetheless, both the severity and duration of shock are typically adjusted to align with specific research objectives (3). Reported MAP targets across studies range from 20 to 48 mmHg, with the most commonly applied values falling between 30 and 35 mmHg. Shock duration also varies substantially, with experimental protocols employing timeframes ranging from 30 to 90 minutes. The fixed-pressure model is limited to short durations, as decompensation and death typically occur within 60–90 minutes at the typically selected pressure values (MAP 25–38 mmHg), regardless of blood reinfusion or fluid resuscitation (26). Prolonged maintenance of such low pressures leads to irreversible organ injury and rapid physiological decline. Consequently, this model is suitable for investigating early shock mechanisms but not for assessing long-term outcomes or late-stage interventions (74).

Another key methodological variable is the rate of blood withdrawal: while some studies employ rapid exsanguination techniques (75), others utilize slower, controlled withdrawal over extended periods (76), depending on the desired hemodynamic profile and research objectives.

Advantages and limitations: A major advantage of the fixed-pressure hemorrhagic shock model is its high degree of reproducibility and experimental standardization. By maintaining a constant MAP, this model allows precise control over shock severity and facilitates comparisons across studies. One limitation of the fixed-pressure model is that maintaining a constant target MAP, may attenuate some early compensatory responses, such as sympathetic activation, peripheral vasoconstriction, and redistribution of blood flow that naturally occur during uncontrolled hemorrhage. Consequently, isobaric models may not fully capture the complexity of real-life hemorrhagic shock (3).

Applications: The fixed-pressure HS model is widely used to investigate the dysregulated inflammatory response associated with blood loss. This constant-pressure approach serves as a valuable pathophysiological tool for studying hemorrhage-induced organ and tissue injury, microcirculatory disturbances, and alterations in acid-base balance. Additionally, the fixed-pressure model provides a controlled platform for evaluating new resuscitation strategies and testing potential therapeutic agents. Owing to its high degree of standardization, it has been extensively adopted in preclinical research across a range of investigative domains, from immunology to critical care medicine (11).

Fixed-volume hemorrhagic shock

In the fixed-volume HS model, a predetermined volume of blood-typically calculated as a percentage of the animal’s body weight or total circulating blood volume-is withdrawn to induce shock. Following hemorrhage, a defined period is allowed for spontaneous physiological compensation. Importantly, blood pressure is not actively maintained during the shock phase, distinguishing this approach from pressure-controlled models (7). Although the procedure can be performed without catheterization, animals are usually anesthetized and catheterized to enable precise blood withdrawal, physiological monitoring, therapeutic interventions, or subsequent resuscitation. The volume of withdrawn blood varies across studies, ranging from 30% (77), 40% (78, 79), 45% (80, 81), 50% (82), 55% (83) to as much as 60% of total blood volume (84). Similarly, the duration of the shock period is highly variable, from brief exposures of 30 (85) or 40 minutes (83) to extended durations of up to 6 hours (81), depending on the study design and research objectives.

Estimated blood volume (EBV), also referred to as total blood volume (TBV), is a fundamental parameter for standardizing hemorrhagic shock protocols. Several formulas are commonly used to estimate EBV based on the body weight of the experimental animal, most frequently rodents. A widely accepted approach involves using a fixed conversion factor per gram of body weight. For rats, EBV is typically estimated as 6–7% of total body weight (TBW), resulting in the formula: EBV = 0.06–0.07 × body weight [g]. Some studies employ more specific formulas, such as EBV [ml] = (body weight [g] × 0.06) + 0.77, which accounts for non-linearities in blood volume distribution (86). To facilitate cross-study comparisons and minimize inconsistencies arising from different EBV estimation formulas, it is recommended to express hemorrhage volume not only as a percentage of EBV but also in absolute terms (ml/kg body weight). For example, in rats, the estimated blood volume corresponds approximately to 60–70 ml/kg. Reporting blood loss relative to this value helps ensure consistency and clarity across experimental models. In larger animals, correction factors may be applied to adjust for fat content or age-related physiological changes, as the blood volume-to-body weight ratio decreases with increasing body size. Accurate estimation of EBV is critical for determining the percentage of blood loss, controlling the severity of hemorrhagic shock, and enabling reliable comparisons across experimental models.

Advantages and limitations: An advantage of the fixed-volume hemorrhage model is its ability to elucidate the animal’s innate compensatory mechanisms in response to a defined volume of blood loss. This model facilitates the study of acute physiological adaptations to a controlled and standardized hemorrhagic event. However, the conversion between blood volume and body weight - both across species and among individuals within the same species - can compromise reproducibility. Consequently, this model offers less experimental standardization compared to the fixed-pressure approach (3).

The primary strength of the fixed-volume approach lies in its utility for investigating hemodynamic responses and compensatory mechanisms following acute blood loss. Nonetheless, the degree of hypotension is not precisely controlled, which limits the precision with which its effects can be assessed and reduces experimental reproducibility. Additionally, in rats, the blood volume-to-body weight ratio decreases linearly with increasing animal weight (100–400 g), as larger animals have relatively more adipose tissue and less circulating blood volume. This variability can significantly influence experimental outcomes, making strict weight control during animal selection and experimental design essential (82).

Applications: According to the Advanced Trauma Life Support (ATLS) classification, Class IV hemorrhage is defined as blood loss exceeding 40% of total circulating volume and is associated with a mortality rate of over 30% in clinical settings (87). To replicate this critical physiological state in preclinical research, many investigators adopt fixed-volume protocols that simulate severe hypovolemia and allow systematic evaluation of fluid resuscitation strategies and pharmacologic interventions (88).

This model is frequently applied in survival studies, where therapeutic efficacy is assessed based on physiological recovery and post-shock survival outcomes (83). It provides a reliable platform for assessing metabolic and cellular responses to hypovolemia, including alterations in carbohydrate metabolism, blood glucose levels, hepatic glycogen depletion, anaerobic glycolysis, and lactate accumulation (54). These parameters are critical indicators of tissue hypoxia, impaired perfusion, and energy failure - all hallmarks of hemorrhagic shock progression.

The fixed-volume model also allows detailed evaluation of histopathological changes in key organs, including the lungs, liver (89), and intestines (90). Common findings include tissue necrosis, microvascular congestion, inflammatory infiltration, and endothelial injury, reflecting the systemic impact of hemorrhagic insult (89, 90). The quantification of these structural and biochemical endpoints makes the fixed-volume model particularly useful for assessing the efficacy of therapeutic strategies aimed at restoring perfusion, preserving organ function, and limiting ischemia-induced tissue damage.

To facilitate direct comparison, the principal methodological characteristics and distinguishing features of fixed-pressure and fixed-volume protocols are presented in Table 2.

Table 2. Comparison of controlled hemorrhage shock models: fixed-pressure and fixed-volume.

Combined fixed-pressure/fixed-volume model

In addition to the classical fixed-pressure and fixed-volume paradigms, a hybrid approach integrating both principles have been developed. In this model, blood is rapidly withdrawn until a target MAP is achieved, after which the bleeding rate is adjusted to maintain this pressure while removing a predefined blood volume normalized to body weight. This design reproduces the dynamics of controlled bleeding and partial hemodynamic stabilization typically observed following initial first aid or tourniquet application. Although less standardized across studies, hybrid models have been shown to more accurately replicate the physiological conditions of controlled hemorrhage than either fixed-pressure or fixed-volume models alone. Recent work by Dupas et al. further refined this approach in rats, confirming its feasibility and translational value for investigating cardiovascular and metabolic adaptations during hemorrhagic shock and subsequent resuscitation (74).

Uncontrolled hemorrhagic shock models

In contrast to controlled HS models, uncontrolled hemorrhagic shock (UHS) models are designed to replicate clinically relevant scenarios in which bleeding is not mechanically or pharmacologically restricted. In these models, hemorrhage progresses spontaneously, with its dynamics governed solely by the animal’s physiological hemostatic mechanisms (91). Models of uncontrolled hemorrhage, such as liver laceration, spleen transection or tail amputation, are widely used in preclinical studies to reflect the mechanisms of abdominal trauma and evaluate the efficacy of new hemostatic therapies (3, 14).

The most frequently applied rat models of uncontrolled hemorrhagic shock (UHS) are summarized in Table 3, illustrating their methodological diversity and relevance for translational trauma research.

In certain experimental setups, UHS is incorporated into hybrid protocols, in which an initial phase of controlled hemorrhage (typically fixed-volume) is followed by a trauma-induced, uncontrolled bleeding event. This two-phase approach allows researchers to study the transition from compensated shock to decompensation and the subsequent development of coagulopathy, more accurately reflecting the complex pathophysiology of polytrauma (104).

Table 3. Summary of commonly used uncontrolled hemorrhagic shock (UHS) models.

Advantages and limitations: UHS models facilitate the evaluation of the effectiveness of pharmacological and technological interventions in conditions analogous to clinical settings, particularly in the context of non-compressive torso injuries. Their principal advantage lies in the ability to capture the spontaneous dynamics of bleeding, clot formation, and hemodynamic decompensation, thereby enhancing translational relevance. However, these models are inherently characterized by high variability, arising from anatomical differences and the unpredictable nature of bleeding. This variability can compromise reproducibility and complicate cross-study comparisons (14). Moreover, UHS models do not fully replicate the complexity of human polytrauma, and interspecies differences may limit the translational validity of specific physiological and biochemical parameters. Therefore, results derived from these models should be interpreted with caution and complemented by standardized protocols and adequate sample sizes to account for intrinsic biological variability (7).

Applications: UHS models are used to study hemostasis mechanisms, evaluate the efficacy of hemostatic agents and resuscitation therapies, and examine the development of immunopathies and coagulopathies associated with trauma. They provide a valuable platform for testing novel pharmacologic interventions and hemostatic technologies under conditions of high mortality risk due to uncontrolled bleeding. Moreover, UHS models enable real-time analysis of blood loss dynamics, hemodynamic instability, and the physiological compensatory responses that characterize severe hemorrhage (14).

Traumatic hemorrhagic shock (T/HS) models

T/HS models acknowledge that hemorrhage in clinical settings rarely occurs in isolation; it is almost always accompanied by physical injury to soft tissues, internal organs, and bones. These models are designed to simulate the multifactorial nature of traumatic hemorrhage by integrating mechanical trauma with either controlled or uncontrolled bleeding (11, 108).

Commonly used T/HS models include procedures such as midline laparotomy, femoral fracture, hepatic crush injury, and combined soft tissue-muscle trauma. One widely adopted protocol involves a sequence of injuries, including a midline abdominal incision, blunt liver lobe crush, intestinal manipulation, hind limb muscle contusion, and a closed femoral fracture (90). This approach reflects the complexity of polytrauma observed in clinical scenarios, such as battlefield injuries, motor vehicle accidents, or high-impact falls.

Compared to isolated hemorrhagic models, T/HS protocols elicit stronger and more dynamic inflammatory, immunological, and metabolic responses - factors critically involved in the pathogenesis of secondary complications such as multi-organ dysfunction syndrome (MODS) and sepsis (108, 109). Consequently, T/HS models are highly valuable for investigating interventions aimed at modulating immune function, inflammation, and tissue repair. Successful implementation of T/HS models requires careful control of key experimental variables, including injury type, hemorrhage volume and rate, timing of resuscitation, and overall shock duration. Model selection should be guided by the specific research question and ideally mirror a well-defined clinical trauma scenario (11).

Advantages and limitations: These models enable real-time monitoring of metabolic, immunological and hemostatic changes, as well as the assessment of the impact of various resuscitation strategies, including delayed resuscitation (71). However, T/HS models also have notable limitations, most importantly low reproducibility and limited experimental control. These factors can complicate cross-study comparisons and hinder the generation of robust, generalizable data (91). Such challenges pose significant obstacles to conducting meta-analyses and translating preclinical findings into clinical applications.

Applications: Rat T/HS models are used to study the mechanisms behind organ damage and the inflammatory response. They are also used to study post-traumatic coagulopathy and to evaluate new therapeutic strategies, including drugs, fluid therapy, and surgical interventions (6, 71). Their application is essential for preclinical validation of therapies designed to control hemorrhage, modulate systemic inflammation, and prevent downstream complications, including coagulopathy, sepsis, and MODS (108, 109).

Combined traumatic brain injury (TBI) and hemorrhagic shock models

Traumatic brain injury (TBI) presents a broad spectrum of clinical manifestations, ranging from mild cognitive and emotional disturbances to severe motor deficits and death. Severe TBI is often accompanied by hypotension and cerebral ischemia, key contributors to secondary brain injury that significantly increase mortality risk (108). Among trauma patients, the most common early fatal injury pattern involves the combination of HS and TBI (109), frequently observed in high-energy trauma scenarios such as motor vehicle collisions, falls from significant height, or blast-related combat injuries. Blood loss accounts for nearly a third (~30%) of all trauma-related deaths (110).

The controlled cortical impact (CCI) model, originally developed by Dr. David T. Povlishock and colleagues, is one of the most extensively characterized and widely used experimental paradigms for simulating TBI in preclinical research (111). The CCI model offers high reproducibility and precise control over injury parameters, making it suitable for studying both acute and chronic effects of TBI. Typically, the procedure involves a craniotomy followed by a calibrated impact to the exposed cortex using a pneumatic or electromagnetic device mounted on a stereotaxic frame. Functional outcomes are evaluated through a combination of histological analysis, biochemical markers, and behavioral assessments. Cognitive performance can be assessed using the Morris Water Maze (MWM), Barnes Maze, or Novel Object Recognition (NOR), while motor function is evaluated using Beam Balance, Beam Walking, Rotarod, and Wire Grip tasks (112).

Although the classic CCI protocol involves craniectomy and direct impact to the exposed dura mater, modified variants employing closed-skull impact without craniectomy are increasingly utilized to model mild or repetitive brain injuries that more closely resemble those sustained by athletes and military personnel. In closed-skull models, when the impact parameters (e.g., depth, speed, and tip shape) are selected appropriately, the risk of skull fracture is minimal, and the injury is confined to the brain tissue without any complications involving the bone. This enables repeatable mild or moderate injuries to be induced while maintaining skull integrity, which is crucial for research into the pathophysiology of concussion, repeated injuries, and the long-term consequences of TBI (112).

Apart from the widely used CCI model, other models of traumatic brain injury, such as weight drop injury (WDI) (113), fluid percussion injury (FPI) (114), and blast-induced traumatic brain injury (bTBI) (115), also play an important role in experimental studies combining hemorrhagic shock with brain injury. The WDI model involves dropping a calibrated weight onto the closed skull of an animal, producing injuries of variable severity - typically ranging from mild to moderate - and characterized predominantly by diffuse damage without well-defined focal lesions. The WDI model is particularly useful for studying neuroinflammatory mechanisms, behavioral disorders, and long-term cognitive deficits following mild TBI. It can also be used to assess interactions with hemorrhagic shock in the context of neuroimmunology and omics biomarkers (116, 117).

FPI, in both its lateral (LFPI) (118) and midline (midline FPI) (119) forms, causes injury by applying a brief impulse of fluid pressure to the dura mater. This leads to a combination of focal and diffuse damage, including oedema, hemorrhages, blood-brain barrier disruption, and extensive neuroinflammatory activation. FPI is considered one of the best-characterized TBI models, as it allows precise control of injury severity and integration with proteomic and metabolomic analyses. This is particularly important in studies of the pathomechanisms of hemorrhagic shock with brain injury (120, 121).

Blast-induced traumatic brain injury reflects the injuries caused by shock waves typical of the military population and uses shock wave generators to cause diffuse damage to cerebral vessels, blood-brain barrier disruption, increased oxidative stress, and activation of neuroinflammatory pathways. bTBI is characterized by a unique neuropathological profile, including neuronal apoptosis, oedema, and activation of specific signaling pathways, making this model particularly useful for studying the molecular mechanisms of the early phase of injury and identifying omics biomarkers (115).

Advantages and limitations: The principal advantage of TBI/HS models lies in their enhanced translational relevance: they capture the compounded effects of two major drivers of morbidity and mortality in trauma patients. Such models are particularly valuable for studying systemic inflammatory responses, blood-brain barrier disruption, and coagulopathy. However, they also introduce greater variability and technical complexity. Increased mortality rates, challenges in maintaining consistent injury severity, and difficulties in standardizing combined insults can limit reproducibility. Moreover, anesthetic and resuscitation requirements may themselves confound both neurological and systemic outcomes.

Applications: Importantly, the CCI model can be integrated with both controlled hemorrhage models (93, 122, 123) and uncontrolled hemorrhagic shock models (102, 124, 125), thereby more accurately reflecting the complex injury patterns observed in polytrauma patients. These combined models allow researchers to examine the interplay between systemic hypoperfusion and brain injury, particularly with respect to neurological deficits and the systemic inflammatory response. Integrated TBI+HS models are essential for studying mechanisms of neuroinflammation, blood-brain barrier disruption, oxidative stress, and neurodegeneration. They also provide a valuable platform for testing novel pharmacologic and resuscitative strategies aimed at improving outcomes in patients sustaining multiple traumatic insults.

EXPERIMENTAL OBJECTIVES

The choice of accompanying trauma in hemorrhagic shock models should be guided by the primary experimental aim. If the focus is on early mortality and cardiovascular decompensation, isolated hemorrhage - whether controlled or uncontrolled - is often sufficient, as it reliably induces fatal circulatory collapse. In contrast, when the goal is to study downstream organ dysfunction, inflammatory cascades, or coagulopathic mechanisms, trauma-integrated models (T/HS or TBI+HS) become essential, as the addition of tissue injury profoundly modifies hemostatic, immune, and metabolic responses. Recognizing this distinction is critical for aligning model design with the pathophysiological processes under investigation.

Rat models are widely used not only in research on hemorrhagic shock, but also in the analysis of other serious cardiovascular diseases, such as autoimmune myocarditis. An example is the work of Stasiak et al. (126), in which the EAM model was used to assess the effect of histamine H1–H4 receptor blockade on disease progression and cardiac remodelling, confirming the usefulness of rat models in research on the mechanisms and treatment of cardiomyopathy.

APPLICATION OF OMICS TECHNOLOGIES IN
EXPERIMENTAL MODELS OF SHOCK AND TRAUMA

The integration of omics methodologies as endpoints in experimental models of hemorrhagic shock and trauma provides new opportunities for biomarker discovery, patient phenotyping, and elucidation of underlying pathophysiological mechanisms. Transcriptomic profiling in rat models of HS has identified genes associated with inflammation, oxidative stress, and tissue repair, offering a foundation for the development of novel therapeutic targets (127). Metabolomics facilitates comprehensive analysis of metabolic changes occurring during shock and resuscitation, including disturbances in glycolysis, the citric acid cycle (TCA cycle), lipid metabolism, and amino acid metabolism. This analytical approach enables the identification of metabolic profiles associated with the risk of death and different phases of care (128-130). For instance, as demonstrated by animal models and clinical studies, fluctuations in acylcarnitine, choline, and biogenic amine concentrations have been observed to be associated with shock severity and prognosis (131). The combination of proteomics with metabolomics facilitates the identification of organ damage patterns and the integration of molecular data with clinical parameters, thus enabling more precise patient stratification and personalized therapy (131, 132). From a methodological perspective, it is imperative to employ multiple sampling at varying time intervals, integrate data from diverse omics (transcriptome, proteome, metabolome, and lipidome), and utilize advanced analytical tools to identify phenotypes and key biological pathways. In the future, the validation of omics biomarkers in translational studies and their integration with clinical data may enable the implementation of precision medicine in the care of patients in shock (130).

CONCLUSIONS

Rat models of hemorrhagic shock remain indispensable tools for investigating the pathophysiology of trauma and for evaluating resuscitative/pharmacologic interventions. Controlled models, particularly recent refinements of fixed-pressure and fixed-volume protocols, enable precise standardization of shock severity and improved reproducibility, allowing mechanistic and interventional studies under well-defined conditions. Nevertheless, despite advances in feedback-controlled systems that simulate dynamic bleeding, these approaches still cannot fully replicate the complex autoregulatory processes characteristic of uncontrolled hemorrhage.

Uncontrolled models, although inherently more variable, preserve key physiological and hemostatic dynamics that occur during spontaneous bleeding and thus retain critical translational value. The choice between controlled and uncontrolled paradigms should therefore be guided by the specific scientific aim: mechanistic reproducibility versus clinical realism. Combined and hybrid models, including trauma-associated and TBI+HS paradigms, offer an increasingly relevant bridge between these extremes.

Persistent methodological limitations - including strain-dependent variability, anesthetic effects, and non-standardized endpoints - underscore the need for continued protocol harmonization and transparent reporting. Future progress will depend on integrating controlled precision with clinical authenticity, aided by multi-omics approaches and improved physiological monitoring, to enhance the predictive validity of preclinical hemorrhagic shock research.

Data availability: No datasets were generated or analyzed during the current study.

Conflict of interests: None declared.

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