Traditional hemorrhagic shock (HS) assessment methods focus solely on macrocirculatory indicators, failing to detect microcirculatory disturbances at an early stage, which may lead to misjudgment of disease severity or delayed intervention. This randomized controlled animal experimental study aims to construct a novel three-level quantitative assessment integrating basic vital signs, blood gas indicators, and microcirculatory parameters, to determine its accuracy and reliability in distinguishing mild, moderate, and severe HS, thereby providing a scientific tool for early precise staging and targeted resuscitation in clinical practice.

Following the quality control principles of experimental research (randomization, blinded assessment), 20 experts with associate senior professional titles or above in the fields of anesthesiology, critical care medicine, field surgery and other relevant disciplines were selected to conduct 3 rounds of anonymous Delphi consultations. Shock evaluation indicators were screened and their weights were determined by calculating the expert positive coefficient, opinion concentration degree and opinion coordination degree. A three-level assessment system consisting of basic vital signs, blood gas indicators and microcirculatory parameters was constructed, and quantitative scoring standards were formulated. A total of 64 SPF-grade SD rats (12 weeks old) were randomly divided into 4 groups (n=16): a sham operation group and mild, moderate, and severe HS groups (with blood loss accounting for 20%, 35% and 50% of total blood volume, respectively). Side stream dark-field (SDF) microcirculation imaging was employed to detect microcirculatory indicators, and mean arterial pressure (MAP) and oxygen saturation were recorded simultaneously to validate the accuracy of the assessment method.

The positive coefficients (100.0%, 100.0%, 88.9%) and coordination coefficients (Kendall’s W increased from 0.144 to 0.444, P<0.05) of the 3 rounds of expert consultations were relatively high, with a coefficient of variation ranging from 0.07 to 0.27, indicating a significant improvement in the consistency of expert opinions. Finally, 5 core secondary indicators were identified, with their weights as follows: perfused vessel density (0.1388), MAP (0.1318), oxygen saturation (0.1299), microvascular flow index (0.1281) and perfused vessel proportion (0.1263). The quantitative scoring standard showed that 5 to 10 points indicated mild HS, 11 to 15 points moderate, and 16-20 points severe. In animal experimental validation, the accuracy of this method in distinguishing mild, moderate and severe HS was 93.75% (95%CI: 71.7% to 98.9%), 93.75% (95%CI: 71.7% to 98.9%) and 100% (95%CI: 83.8% to 100%), respectively.

Our novel quantitative HS assessment tool based on SDF technology and Delphi method, can accurately distinguish HS of different severity levels (accuracy rate >90%), effectively integrate the monitoring dimensions of macrocirculation, oxygenation, and microcirculation. Our tool addresses the limitation of traditional assessment tools in neglecting microcirculation, providing reliable assessment for early precise identification, dynamic monitoring of disease progression, and targeted resuscitation of HS, with significant potential for clinical translation.

To characterize the myocardial proteomic and metabolomic alterations induced by high-altitude exposure and hemorrhagic shock (HS) under hypobaric hypoxic conditions, and to evaluate the regulatory effects of early lactated Ringer's (LR) resuscitation on related molecular abnormalities, providing evidence for elucidating the mechanisms of myocardial injury in high-altitude HS and optimizing resuscitation strategies in such conditions.

Forty SPF-grade male SD rats (10 to 12 weeks old, weighing 180 to 220 g) were randomly divided into a normoxic control group (NC), a high-altitude sham group (Sham), a high-altitude uncontrolled HS (UHS) and a high-altitude HS with LR resuscitation group (LR1h), with 10 animals in each group. Hypobaric hypoxic condition was induced by rats placed in a hypobaric chamber to simulate an altitude of 5000 m for 48 h. After chamber exit, the rats received an injection of oleic acid via the tail vein for 0.5 h, followed by splenic artery transection to induce uncontrolled hemorrhage until the mean arterial pressure (MAP) decreased to 40 mmHg. Thus, a high-altitude HS model was established. After successful modeling, the UHS group received no resuscitation with continuous monitoring only, while the LR1h group received an infusion of LR solution via the femoral vein for 1 h resuscitation under hypobaric condition. At designated time points, heart tissues were collected for 4D-FastDIA quantitative proteomics (n=4) and untargeted metabolomics analysis (n=6). Principal component analysis (PCA), differential protein/metabolite screening, and gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were performed to characterize myocardial molecular expression profiles under different treatment conditions.

High-altitude exposure effect (Sham vs NC): totally 229 differentially expressed proteins (DEPs) were identified (154 up-regulated and 75 down-regulated, P<0.05). Enrichment analyses showed up-regulation of immune and inflammatory response-related processes, and downregulation of cardiac contraction, mitochondrial gene expression, and protein synthesis. There were 633 differential metabolites identified (509 up-regulated and 124 down-regulated, VIP≥1, P<0.05), primarily enriched in fundamental metabolic pathways, such as lipid, terpenoid/polyketide, and amino acid metabolic pathways. HS shock effect (UHS vs Sham): A total of 193 DEPs were identified (42 up-regulated and 151 down-regulated, P<0.05), characterized by enhanced cholesterol and lipid metabolism, whereas global down-regulation of complement-coagulation cascades, lysosomal function, immune and inflammatory regulation, and multiple infection-related pathways. Among the 1100 identified differential metabolites, there were 267 up-regulated and 833 down-regulated (VIP≥1, P<0.05). The types of enriched pathways were generally consistent, but the overall number of enriched compounds was decreased, with more pronounced reductions in key metabolic axes, including lipid metabolism (from 31 to 20), terpenoid/polyketide metabolism (from 20 to 14), and amino acid metabolism (from 11 to 6). LR resuscitation effect (LR1h vs UHS): A total of 108 DEPs were identified (39 up-regulated and 69 down-regulated, P<0.05). Vesicular transport, autophagy, and renin-angiotensin system pathways were up-regulated, but complement-coagulation cascade, humoral immunity, inflammatory response, and multiple infection-and metabolism-related pathways remained down-regulated, along with suppression of oxygen transport processes. Totally 494 differential metabolites were identified (456 up-regulated and 38 down-regulated, VIP≥1, P<0.05). Enriched pathways remained concentrated in the above main metabolic routes, but the number of enriched compounds in these pathways further decreased compared with the shock state, including lipid metabolism (from 20 to 10), terpenoid/polyketide metabolism (from 14 to 12), and amino acid metabolism (from 6 to 0).

Under high-altitude hypobaric hypoxia, HS induces myocardial immune/coagulation suppression and metabolic disorder. Early resuscitation with LR solution fails to reverse these molecular alterations, indicating that simple crystalloid volume resuscitation is insufficient to protect the myocardium in high-altitude hemorrhagic shock.

To investigate the protective effect of mitochondrial fission inhibitor 1 (Mdivi-1), on organ function in rats with explosive blast injury combined with hemorrhagic shock.

A total of 192 SD rats (half male and half female, 12 weeks old, weighing about 220 g) were randomly divided into 6 groups: Sham group (only surgical incision along the midline of the abdomen), model group (ESH group, thermal radiation and shock wave injury followed by femoral artery hemorrhage), lactated Ringer’s solution resuscitation group (ESH+LR group, LR solution infusion in the femoral vein for resuscitation), and low-, middle- and high-dose Mdivi-1 groups (0.1, 0.5 and 1.0 mg/kg Mdivi-1 intervention after infusion of LR solution). Fluorescent protein tracing was used to determine the leakage amount of fluorescent protein in the lung and kidney tissues to evaluate the vascular permeability. Evans blue dye staining was employed to observe the intestinal permeability and pulmonary vascular permeability. Laser Doppler flowmetry was applied to monitor the tissue blood perfusion in the liver, kidneys, and intestine. Serum levels of cardiac injury marker troponin I (TNI), liver function markers aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and renal function markers serum creatinine (Scr) and blood urea nitrogen (BUN) were detected to evaluate the functions of corresponding organs. The water contents of the lungs and brain were calculated by measuring wet weight and dry weight of the lung and brain tissues. Blood pressure, heart rate, and respiratory rate were monitored. The survival time and 72-hour survival rate were recorded and calculated.

Compared with the Sham group, the ESH group exhibited significantly increased vascular permeability in the lungs and kidneys as well as intestinal tissue (P<0.05), along with obviously elevated water contents in the lungs and brain (P<0.05), and decreased blood perfusion in the liver, kidneys, and intestine by 57.1%, 39.2%, and 43.2% of the Sham group, respectively (P<0.05), elevated levels of TNI, AST, ALT, Scr and BUN (P<0.05), mean survival time of 3.8±1.1 h, and a 72-hour survival rate of 0 (P<0.05). Although LR solution resuscitation reduced vascular permeability and alleviated organ injury in rats with explosive injury combined with hemorrhagic shock, there were no significant differences compared to the ESH group (P>0.05). Mdivi-1 treatment notably decreased vascular permeability in the lungs and kidneys and intestine, and water contents in the lungs and brain when compared with the LR group (P<0.05), with the dose of 0.5 mg/kg demonstrating the most significant effect. Additionally, Mdivi-1 treatment also significantly enhanced organ perfusion, improved organ functions, prolonged survival time, and increased survival rate. The 0.5 mg/kg treatment resulted in a 72-hour average survival time 55.64 h and a survival rate of 62.5%.

Mitochondrial fission inhibitor Mdivi-1 can reduce the permeabilities in the lungs, kidneys and intestine, improve tissue blood perfusion, protect the organ functions of the heart, liver and kidneys, and finally prolong survival time and increase survival rate in rats with concomitant explosive blast injury and hemorrhagic shock.

To systematically investigate the metabolic characteristics of hemorrhagic shock (HS) under simulated high-altitude conditions by integrating metabolomics and machine learning algorithms, and screen the biomarkers for predicting HS under these conditions.

Eighty SPF-grade male SD rats (10 to 12 weeks old, weighing 180 to 220 g) were randomly divided into a sham operation group (Sham) and an uncontrolled HS group (UHS), with 40 animals in each group. All rats were placed in a hypobaric hypoxia chamber to simulate an altitude of 5000 m for 48 h. After removal from the chamber, the rats were given an injection of oleic acid immediately via tail vein, and then in 30 min latter, those from the UHS group were induced to free bleeding by splenic artery transection, with the endpoint set at a mean arterial pressure (MAP) of 40 mmHg. Thus, a rat model of HS under stimulated high-altitude conditions was established. Metabolomic analysis was performed on the serum samples to identify differential metabolites between the Sham and UHS groups. Weighted gene co-expression network analysis (WGCNA) was carried out to identify metabolites associated with UHS. Three machine learning methods, including least absolute shrinkage and selection operator (Lasso) regression, random forest (RF), and support vector machine-recursive feature elimination (SVM-RFE) were employed to identify relevant biomarkers for HS at high-altitude. Based on 10-fold cross-validation, the diagnostic performance of the biomarkers was evaluated with receiver operating characteristic (ROC) curve analysis, and the area under the curve (AUC) was calculated.

Principal component analysis (PCA) of the metabolomic data showed clear separation between the samples from the Sham and UHS groups. Orthogonal partial least squares-discriminant analysis (OPLS-DA) further confirmed significant differences in metabolic profiles between the 2 groups. There were 5398 metabolites identified, with the UHS group having 391 metabolites significantly down-regulated (VIP > 1, FC < 1/1.5, P < 0.05) and 1181 metabolites obviously up-regulated (VIP > 1, FC > 1.5, P < 0.05) when compared to the Sham group. Among the metabolites, the most altered metabolites were lipids, including FFA (18:2), FFA (18:1), FFA (12:0), FFA (14:0), ergosterol, mesaconic acid, suberic acid, gamma-linolenic acid, and PC [22:5(4Z, 7Z, 10Z, 13Z, 16Z)/22:6(4Z, 7Z, 10Z, 13Z, 16Z, 19Z)], and then followed by organic acids and derivatives, such as methionine, citrulline, creatine, L-lactic acid, oxalacetic acid, and 2-acetolactate. Pathway enrichment analysis showed that these differential metabolites were mainly involved in glutamine metabolism, taurine metabolism, alanine, aspartate and glutamate metabolism, as well as glycerophospholipid metabolism. WGCNA analysis found that MEturquoise module exhibited the strongest positive correlation with UHS (R=0.95, P=8e-40), while the MEsalmon module showed the strongest negative correlation with UHS (R=-0.67, P=2e-11). With thresholds of |Gene Significance (GS)| > 0.2 and |Module Membership (MM)| > 0.8, 344 characteristic metabolites were identified, and all of them were significantly associated with HS in a high-altitude environment. The 3 machine learning algorithms yielded 3 biomarkers, that is, methionine, D-inositol-4-phosphate and phosalone. Significantly increased D-inositol-4-phosphate and phosalone while decreased methionine were observed in the UHS group than the Sham group. ROC curve analysis revealed that the AUC values of D-inositol-4-phosphate, phosalone and methionine were 0.988, 0.950 and 0.988, respectively, indicating that the 3 biomarkers having good predictive efficiency for HS under simulated high-altitude condition.

HS rats under simulated high-altitude conditions present significantly disturbed metabolic profiles, characterized by substantial changes across multiple pathways. The metabolites D-inositol-4-phosphate, phosalone and methionine may serve as potential biomarkers for predicting the occurrence or evaluating the severity of HS in high-altitude environments.

To establish lethal shock models in high-altitude, high-cold, and high-heat environments based on the conventional environment hemorrhagic shock (HS) model established in our laboratory, in order to provide animal models for lethal shock treatment in special environments.

A total of 864 SPF-grade male SD rats (12 weeks old, weighing 200±20 g) were divided into high-altitude environment group (simulated 4000 m altitude in a low-pressure oxygen chamber, exposed to 22±2 ℃ for 48 h), high-cold environment group (altitude 200 to 300 m, exposure to -20 ℃ for 6 h), high-heat environment group (altitude 200 to 300 m, exposure to 35 ℃ for 12 h), and conventional environment control group (no environmental pretreatment) according to different environmental pretreatment, with 216 animals in each group. After environmental pre-conditioning, 4 types of spleen injury methods were used to induce free intraabdominal bleeding to construct corresponding uncontrolled HS model. The injury methods were as follows: ① Transverse transection at 3 cm away from the end of the splenic parenchyma; ② Transverse transection separately at 3 and 5 cm away from the end of the splenic parenchyma; ③ Transverse arteriotomy of a small arterial branch at the splenic tail; ④ Transverse arteriotomy of a small arterial branch at the splenic tail combined with transection at 3 cm away from the end of the splenic parenchyma. Free intra-abdominal bleeding was induced by above 4 methods until the mean arterial pressure (MAP) dropped to 40 mmHg, which was defined as successful establishment of the uncontrolled HS model. After successful modeling, lactated Ringer's (LR) solution was employed to maintain MAP at 50 to 60 mmHg at low pressure for 1 h. After the splenic artery was ligated for complete hemostasis, definitive resuscitation was obtained with LR solution of 2×blood loss volume. MAP, respiratory rate (RR) and heart rate (HR) were monitored, cardiac function [troponin I (TnI)], liver function [aspartate aminotransferase (AST), alanine aminotransferase (ALT)]、and renal function [blood urea nitrogen (BUN), serum creatinine (Scr)] were evaluated, survival time was observed, and 6-hour survival rate was calculated to screeen the appropriate modeling methods for each environment. Then the obtained modeling methods were applied on Bama pigs to establish HS models for high-altitude, high-cold, and high-heat environments, respectively. After modelling, the pigs were resuscitated according to the aforementioned treatment regimens, and basic physiological indicators (MAP, RR, HR), organ functions (heart, liver, kidney), and survival status were observed to verify the stability of the modeling methods.

There were differences in the appropriate modeling methods for different environments: the conventional environment preferred method ④, the high-altitude environment preferred method ③, the high-cold environment preferred method ①, and the high-heat environment preferred method ②. All the above methods met the model standards of a 6-hour mortality rate > 70% after shock, a blood loss > 50% after low-pressure resuscitation, and a survival rate > 50% after definitive resuscitation. In the state of shock, the MAP of rats was significantly reduced in each group (P < 0.001), RR and HR were abnormally changed, the indicators of cardiac, liver, and renal functions (TnI, AST, ALT, BUN, Scr)(P < 0.05) were significantly increased, and the survival time was obviously shortened. After LR low-pressure resuscitation combined with definitive resuscitation, the MAP was notably recovered (P < 0.01), the organ function indicators were significantly improved, and the survival time was greatly prolonged. The verification results in Bama miniature pigs showed that in the shock group, MAP was decreased to approximately 40 mmHg, RR was significantly reduced, HR was increased, the 6-hour survival rate was 0%, and the indicators of cardiac, liver, and renal functions (TnI, AST, ALT, BUN, and Scr) were notbly elevated (P < 0.001). In the resuscitation group, the MAP was significantly increased to 58.6 to 68.4 mmHg (P < 0.01), the 6-hour survival rate reached 50.00% to 56.25%, and organ functions were significantly improved. All these findings confirmed the reliability of each model.

Lethal shock models under high-altitude, high-cold, and high-heat environments are successfully established, respectively, and the appropriate standardized modeling methods are identified for each environment. The modelling methods are further verified on Bama miniature pigs, and the constructed models have advantages of stability and reliability for laboratory requirements.

To determine the effects of seawater immersion at 21 ℃ on survival, organ functions, tissue metabolism and homeostasis in rats with hemorrhagic shock.

A total of 128 healthy male SD rats were randomly divided into 4 groups: normal control group (NC), 40% hemorrhagic shock group (HS), normal rats with 21 ℃ seawater immersion (2 h) group (SI), and 40% hemorrhagic shock rats combined with 21 ℃ seawater immersion (2 h) group (HS+SI). The survival rate in each group was observed for 72 h in 2 h after modeling, the changes of cardiac function [cardiac troponin T (cTnT)], liver function [alanine transaminase (ALT) and aspartate aminotransferase (AST) levels], renal function [blood urea nitrogen (BUN) and serum creatinine (SCr) levels], tissue metabolism and internal environment were detected in each group.

As compared with the NC group, the plasma osmolality (OSM), concentrations of Na⁺, K⁺, Cl^- and water contents in the lung and brain were increased, while the Na⁺/K⁺-ATPase activity in the lung and brain was decreased in the HS and SI groups. With the elevation of the damage indexes in heart, liver and kidney functions, the mortality was increased as well in the HS and SI groups. Combination of 21 ℃ seawater immersion resulted in more severe damages induced by simple 40% hemorrhagic shock, shown as further increases in plasma OSM, Na⁺, K⁺ and Cl^- concentrations, and water contents of the lung and brain, as well as further declined Na⁺/K⁺-ATPase activity in the lung (3.81±0.30 U/mg) and brain (7.17±0.51 U/mg) (P<0.05). Serious deterioration was observed in the functions of the heart, liver and kidney, with levels of cTnT, ALT, AST, BUN and SCr elevated to 7.25±0.66 μg/L, 118.60±13.29 U/L, 455.95±46.76 U/L, 19.74±2.12 mmol/L and 56.73±4.08 μmol/L, respectively (P<0.05). The survival rate was reduced to 18.75%, and the median survival time was shortened to 10.50 h in the rats of the HS+SI group.

Seawater immersion at 21℃ exacerbates disorders of the internal environment and tissue metabolism in 40% hemorrhagic shock rats, leading to aggravated multiple organ injury and thus increased mortality.

To observe the early therapeutic effects of a mitochondrial fission inhibitor, mitochondrial division inhibitor 1 (Mdivi-1), on the traumatic shock rats at high altitude.

SD rats were rapidly transported (airlift) from the plain (Chongqing) to the plateau (Lhasa, 3 600 m altitude). After 72 h, the traumatic shock model of rats at high altitude was prepared by free bleeding from splenic artery disconnection until mean arterial pressure (MAP) fell to 40 mmHg. The animals were divided into 5 groups: sham-operation group, shock group, Lactate Ringer's (LR) solution control group, and LR combined with 0.1 and 0.5 mg/kg Mdivi-1 groups. Then the experiments were designed as 2 parts. The rats in the first part were resuscitated with mere LR solution or combined with 0.1 or 0.5 mg/kg Mdivi-1 without hemostatic treatment. With the MAP maintained at 50-60 mmHg, the changes of MAP, bleeding volume, fluid infusion volume and survival rate were observed in each group. In the second part, bleeding control was performed by ligation of the spleen artery after 1 h of low-pressure resuscitation, and definitive resuscitation was continuously given. The changes of blood loss, infusion volume, organ functions, tissue oxygen partial pressure, lung and brain water content and animal survival were subsequently investigated.

When LR solution was used for low-pressure resuscitation without hemostatic treatment, the MAP of rats was maintained at 50-60 mmHg for about 1 h, and then began to drop gradually rather than rise after increasing the amount of fluid input, with the bleeding volume soared obviously. Whereas Mdivi-1 combination treatment maintained MAP at 50-60 mmHg for about 3 h, and significantly reduced the bleeding volume and fluid infusion. Further studies showed that in the hemostatic resuscitation treatment, the rats of the LR control group still had a rather low level of tissue oxygen partial pressure, with elevated water contents in the lung and brain, and seriously impaired functions of vital organs such as the heart, liver and kidney. However, Mdivi-1 (0.1 or 0.5 mg/kg) treatment remarkably improved the damage, increased the tissue oxygen partial pressure, reduced the water contents in the lung and brain, alleviated the impairment of the heart, liver and kidney, and greatly prolonged the survival time of shock animals.

Mdivi-1 is suitable for the early treatment of traumatic shock at high altitude, which can improve organ functions and extend the golden time of treatment.