Traumatic injury is a major global cause of death in all age groups and especially in young adults.1,2 In the United States between 1999 and 2017, more than 2.3 million deaths were ascribed to injuries and the trend is increasing.2,3 importantly, posttraumatic hemorrhagic shock (HS) is the leading cause of preventable death in civilian and combat trauma with the majority occurring before patients reach surgical or critical care facilities.4–6 Traditional resuscitation strategies based on aggressive crystalloids have now been largely replaced by permissive hypotension, or low-volume resuscitation (LVR), with isotonic crystalloids in conjunction with prompt administration of blood products when available. in addition to restoring oxygen-carrying capacity (OCC) and coagulation factors with blood components, minimizing crystalloids reduces the risk of worsening acidosis and dilutional coagulopathy that can exacerbate uncontrolled hemorrhage. Furthermore, excess crystalloids can contribute to acute respiratory distress syndrome, abdominal compartment syndrome, and/or anasarca.7–10 The current “gold standard” involves a balanced transfusion of red blood cells (RBCs), plasma, and platelets, at equivalent ratios; and prehospital blood transfusion has been shown to benefit survival.11,12 However, logistic constraints often limit the availability of blood products soon after injury, when early effective resuscitation is key to survival.4,5,13 Furthermore, low volume crystalloid solutions containing hypertonic saline, hydroxyethyl starch, or other polymers as oncotic volume expanders have shown suboptimal clinical results.14–16 Consequently, there remains a need for a safe and effective solution that is environmentally stable, abundant, and transportable to the prehospital setting.
Of significance, understanding the causal pathophysiology of shock and resuscitation injury may help identify potential targets for treatment. After traumatic HS, inadequate tissue oxygenation and loss of cellular bioenergetics reduce energy-dependent cell volume control mechanisms including the NA+/K+ ATPase pumps. This results in metabolic cell and tissue swelling that compresses the microcirculation, which further hinders organ perfusion and exacerbates ischemic injury. The resulting “no-reflow” phenomenon is further exaggerated by crystalloid overloading with sodium and water,17–22 which promotes a vicious cycle.
In recent years, our lab has demonstrated that isotonic crystalloid [lactated Ringer (LR)] containing 10% polyethylene glycol with a molecular mass of 20,000 Dalton (PEG-20k) is very effective for LVR in acute animal models of severe HS. Due to unique molecular properties (23), PEG-20k exerts a hybrid impermeantoncotic effect targeting the ischemia-related cell-swelling by moving isotonic fluids out of the cells, into the interstitium, and then to the intravascular compartment. This decompresses the microcirculation, reloads the capillaries, and restores local tissue perfusion in an otherwise severely shocked state.17,18,22,23 We previously reported superior outcomes of LVR with PEG-20k compared to saline and LR (crystalloids) and to albumin and hextend (colloids) in acute rodent and/or porcine models of HS.18,23,24 PEG-20k was associated with many fold improvements in tolerance to the low volume state with significant reductions in oxygen debt despite sizable decreases in OCC caused by auto-hemodilution. In light of these findings, we raised the question whether restoring OCC with blood transfusion should remain a major priority of early resuscitation strategies or whether prompt protection of the microcirculation from the detrimental cascade of exchange failure (caused by metabolic cell-swelling) should be the primary target in the early stages of shock treatment. Additionally, evidence is still lacking on whether or not LVR solutions targeting this mechanism are sufficient for survival beyond a few hours of HS, without definitive resuscitation with blood. This is of paramount importance in the prehospital setting with prolonged field care, extended transport time, and in the unusual but dire case of mass casualties.25 Therefore, the aim of this study was to compare the early post-resuscitation and the 24-hour outcomes of LVR with PEG-20k, whole blood (WB), or Hextend, in a preclinical large animal model of life-threatening HS and soft tissue injury.
Animals and Surgical Preparation
This study was approved by the Virginia Commonwealth University's Institutional Animal Care and Use Committee. The experimental design is depicted in Fig. 1. Fasted male Yorkshire pigs (n = 17, Archer Farms, Darlington, MD) weighing 34.8 ± 3.1 kg were sedated with intramuscular ketamine (20 mg/kg) with xylazine (2 mg/kg) followed by anesthesia induction using intravenous propofol (2–3 mg/kg). Anesthesia was maintained with isoflurane at 1%–2% in room air on mechanical ventilation adjusted to an end-tidal CO2 of about 40 mm Hg. A circulating water-warming pad was used to maintain normothermia. Both superficial femoral arteries were cannulated for blood pressure and heart rate monitoring (Power-Lab, ADInstruments inc., Dunedin, New Zealand) and for controlled arterial hemorrhage. Additionally, the external jugular vein was cannulated for fluid administration. A laparotomy and splenectomy were performed to produce soft tissue injury and to mitigate the effect of autotransfusion.
An intravenous fluid loading bolus of LR solution (10 mL/kg) was administered over 10 minutes before baseline data and labs were obtained. Controlled hemorrhage was then initiated by arterial bleeding at a 2 mL/kg rate using a peristaltic roller pump (Masterflex, Cole-Parmer, Chicago, IL) until mean arterial pressure (MAP) reached 35–40 mm Hg. After allowing the animal to compensate to a MAP of 45–50 mm Hg, bleeding was resumed until 1 of the 2 shock endpoints was reached: (1) A plasma lactate of 7.5–8.5 mmol/L was reached within 112 minutes of hemorrhage time and total hemorrhage volume of ≤53% (TBV) or (2) Both hemorrhage time and hemorrhage volume limits were met without achieving the lactate goal.24
LVR and Study Outcomes
Once the shock endpoint was reached, the animals were randomized to receive an intravenous bolus (over 5 minutes) equal to 10% TBV (LVR) of either of PEG-20k, WB, or Hextend (n = 6 each). During hemorrhage, blood was stored in a Viaflex plastic bag containing sodium citrate and was used for resuscitation in animals randomized to the WB group. Vital signs, lactate, and hemoglobin levels were measured every 15 minutes after LVR. The experiment was terminated and the animals euthanized when MAP consistently dropped below 30 mm Hg. If the pigs survived for 240 minutes after LVR, they were recovered from anesthesia. Surviving animals were weaned off anesthesia, recovered, provided postoperative analgesics (Buprenorphine SR), and allowed access to food and water. On postoperative day 1 (POD1), a neurologic assessment was performed followed by a terminal data and specimen recovery procedure.
The primary outcomes of the study were 24-hour survival rate and neurological deficit scoring (NDS). Secondary outcomes included cardiovascular variables (MAP and Heart Rate), oxygen debt indexed by plasma lactate, and labs. Neurologic function on POD1 was evaluated using a standardized scoring that considers behavior and level of consciousness, breathing pattern, cranial nerve function, and motor and sensory function. A NDS of 0–40 is considered as absence of neurologic deficit, a NDS of 400 as brain death.26 An indicator dilution method using hemoglobin estimated changes in the intravascular compartment volume after LVR, assuming no further blood loss was allowed after reaching the controlled hemorrhage endpoint. Several indicator-dilution techniques have been used to calculate intravascular volumes in clinical and experimental settings27,28 and we recently report validation of the hemoglobin method used here against fluorescein isothiocyanate-labeled albumin in a rat model of controlled HS.18 Finally, microvascular perfusion in the distal ileal and sublingual mucosa was measured using orthogonal polarization spectral imaging with the CapiScan device (KK Research Technologies Limited, Devon, UK). Proportion of perfused vessels was calculated as described29,30 to quantitate microvascular perfusion.
Data are expressed as means ± standard deviation. IBM SPSS Statistics for Windows, Version 25.0 (IBM Corp., Armonk, NY) was used for statistical analysis. Comparisons between the 3 groups were performed with Fisher exact test (for categorical variables) and 1-way analysis of variance with Bonferroni multiple comparison correction (for continuous variables). Paired comparisons within each group were performed using paired t-tests. Statistical significance was determined if P < 0.05.
At baseline, animals in all groups had comparable weigh, MAP, lactate, hematocrit, creatinine, and other measures of organ function (Table 1). The overall mean percent hemorrhage (of TBV) was 47.7 ± 6.5, which resulted in an elevation of plasma lactate to 7.9 ± 1.1 mmol/L over an average shock time of 71.7 ± 27.8 minutes. There were no differences between groups in any of these shock parameters before the start of LVR after hemorrhage (Fig. 2).
TABLE 1 -
Baseline Information and 24 h Survival Rates and Neurologic Deficit Scores After Low Volume Resuscitation
||Hextend N = 5
||Whole blood N = 6
||PEG-20k N = 6
||36.6 ± 4.2
||34 ± 2.1
||34 ± 2.6
|Baseline MAP (mm Hg)
||84.8 ± 9.6
||86.2 ± 11
||87.2 ± 14.6
|Baseline lactate (mmol/L)
||2.3 ± 1.1
||1.8 ± 0.5
||2.2 ± 0.7
|Baseline hematocrit (%)
||28.9 ± 1.2
||28.9 ± 2.1
||29.8 ± 1.6
|Baseline creatinine (mg/dL)
||1.2 ± 0.2
||1.1 ± 0.1
||1.2 ± 0.2
|LVR time (min)
||69 ± 73.9
||92.5 ± 104.5
||240 ± 0
|24-h survival rate N (%)
|Survival time (h)
||2.2 ± 1.4
||5.2 ± 9.2
||24 ± 0
|24-h neurologic deficit score
|Overall performance category (OPC):
| OPC 1: normal
| OPC 2: moderate disability
| OPC 1: severe disability
| OPC 1: coma
| OPC 1: brain death or death
P-values are for the 3-group comparison (1-way ANOVA).MAP indicates mean arterial pressure; NS, not significant; PEG-20k, polyethylene glycol 20,000 Daltons.
All animals (100%) in the PEG-20k group survived 24 hours compared to only 1 (16.7%) in the WB group and none (0%) in the Hextend group (P= 0.001). Consequently, mean survival time was substantially longer in the PEG-20k group (24 hours) compared to the 2 other groups (P < 0.001 for both comparisons, whereas it did not differ statistically between the WB and Hextend (5.2 ± 9.2 vs 2.2 ± 1.4 hours, respectively, P= 0.097). The NDS of the 1 surviving pig in the blood group indicated moderate disability whereas all PEG-20k-resuscitated animals had a normal NDS (Table 1 and Fig. 3).
After resuscitation, MAP increased to higher levels with PEG-20k compared with WB and Hextend (P < 0.05). These significant differences were observed throughout the acute phase of the experiment (LVR-15 to LVR-240). Again, no statistical differences were observed in blood pressure between the WB and Hextend groups (Fig. 4). LVR with PEG-20k resulted in a complete lactate clearance by the LVR-240 time point. Normal lactate was maintained for 24-hours despite a decrease in MAP from 75.8 ± 9.1 at LVR-240 to 61.3 ± 9.1 mm Hg at POD1 (P= 0.039). Lactate at LVR-240 (terminal) was 2.9 ± 1 mmol/L which was not significantly different than baseline or POD1 levels. The 1 animal that survived 24 hours in the WB group had a terminal lactate level of 7.4mmol/L. Lactate clearance, and MAP improvement, did not continue in the animals resuscitated with WB and Hextend beyond 1–2 hours. Terminal lactate was not different between the 2 groups (11.3 ± 3.1 and 12.5 ± 4.7, respectively) but they were significantly higher than the terminal lactate in the PEG-20k group (2.9 ± 1 mmol/L P= 0.001).
Significant reduction in hemoglobin concentration was observed 15 minutes after LVR with PEG-20k and throughout the 24-hour study (P < 0.01). Hemoglobin decreased from 9.3 ± 0.7 g/dL at the end of shock to 5.9 ± 0.9 g/dL at LVR-240 (P < 0.001). POD1 hemoglobin was 6.5 ± 1.3 g/dL which was not significantly different than the levels measured at LVR-240. Intravascular volume, which similarly decreased in all groups to an average of 51.1% at the end of HS, was restored to 99% ± 4.6% of baseline volume 30 minutes after LVR with PEG-20k. This volume expansion was maintained for 3.5 additional hours. On the other hand, post-LVR intravascular volume peaked 15 minutes after LVR with WB and Hextend to 57.5% ± 5.5% and 66.2% ± 10.5%, respectively. These increases were not statistically different between the 2 groups but significantly lower than the PEG-20k group (P < 0.001, Figs. 5 and 6).
Changes in microvascular perfusion in distal ileal and sublingual mucosa are shown in Fig. 6B. Perfusion significantly dropped below 50% of baseline after shock immediately before resuscitation. This defect was normalized 30 minutes after LVR with PEG-20k but not with the vehicle volume control (LR).
To our knowledge, this is the first preclinical study to report superior hemodynamic, metabolic, and 24-hour survival outcomes with a non-sanguineous LVR solution compared to WB. All animals resuscitated with PEG-20k after severe class IV hemorrhage survived 24 hours and had a normal NDS, indicating full neurological recovery, whereas none of the animals in the Hextend group and only 1 out of 6 in the WB group survived more than 4 hours after LVR. Our results strongly suggest that improving OCC after severe HS with RBC-rich solutions may not be the most crucial target for early resuscitation. Importantly, PEG-20k and WB target 2 different mechanisms related to HS and resuscitation injury. One is the microcirculatory exchange failure due to cell swelling from the loss of energy-dependent mechanisms responsible for cellular volume regulation (PEG-20k). The second is the OCC depletion directly related to the loss of RBCs during hemorrhage. Despite the increasing emphasis on early blood product administration, there is a large body of evidence from observational studies to suggest that prioritizing the normalization of other elements of shock-induced derangements over OCC may be advantageous. Hemostatic resuscitation with a high plasma:RBCs ratio has recently been advocated for posttraumatic HS as data from combat and civilian studies showed potential survival benefits.31–34 A multicenter prospective study of adult trauma patients who received at least 1 and 3 units of RBCs in the first 6 and 24 hours after admission, respectively (n = 905), found that higher ratio of plasma and platelet to RBCs was associated with decreased mortality within the first 24 hours. Similar findings were reported in a retrospective analysis of the US military population serving in Iraq and Afghanistan between 2003 and 2012 and receiving at least 1 transfusion product within 24 hours of admission.32
On the other hand, the importance of the microcirculation in hemorrhagic and septic shock has been increasingly recognized with more evidence showing startling disparities between macro- and microcirculatory measures, especially in the early hours of shock. This led to the ongoing efforts to develop microcirculation-related indices to identify patients at risk of impeding deterioration which may not be recognizable using traditional monitoring methods, especially in the intensive care unit.17,18,21,35–39 In a recent study of 58 patients with HS who received an average of 6 units of packed RBCs at 3 major trauma centers in the UK, Hutchings et al found that patients who developed multi-organ dysfunctions had a significantly lower perfused vessels density and microcirculatory flow index measured within 12 hours of intensive care unit admission but a similar cardiac index, compared with patients who did not. Therefore, it is not surprising that the perfused vessel density was a more sensitive predictor of multi-organ failure compared to lowest recorded blood pressure and the highest recorded lactate and cardiac index.21 Our study is consistent with this because the microvascular perfusion defect induced by shock was reversed by PEG-20k resuscitation and this solution caused survival and repayment of oxygen debt.
The robust biologic effects of PEG-20k are due to its unique effects at the level of the microcirculation. The polymer targets ischemia-induced cell-swelling that hinders microcirculatory blood flow but does not change OCC in the pre-capillary macrocirculation like WB resuscitation does. PEG-20k is an unconventional polymer that unequally partitions in the microcirculation to establish multiple osmotic gradients for water transfer out of laden parenchymal cells into the capillary space. This response is amplified by its high hydrophilic nature. The reversal of metabolic cell swelling improves microcirculatory exchange and dramatically increases capillary perfusion and oxygen transfer even under the low volume and low OCC states seen in class IV hemorrhage. This is supported by the baseline return of plasma lactate concentrations after PEG-20k resuscitation without addition of more OCC and by the normalization of micro-vascular perfusion imaging outcomes. Some of this hypothesized perfusion effect is supported by higher capillary perfusion pressures from rapid volume expansion and part is supported by decreased resistance to flow by decompression of the tissue capillary beds as ischemic cell swelling is reversed. Our previous studies in rodents18 and this one in pigs demonstrate significant increases in capillary flow after PEG-20k resuscitation in severe shock, relative to the vehicle and volume controls. Comparisons of capillary flow with WB in survival models are not yet available. In the case of OCC-focused resuscitation, RBCs may be largely ineffective because the capillaries remain obstructed by swollen endothelial and parenchymal cells that prevent oxygen transfer. Therefore, these studies provide compelling evidence that it may be more important to restore capillary perfusion than to add more OCC in severe hemorrhage because doing the former restores oxygen delivery to the microcirculation and pays back oxygen debt in the local tissues, even in the face of a 50% drop in OCC after hemorrhage. The corollary is that adding OCC (blood RBC products) after shock may be ineffective if the microcirculation is swollen shut by metabolic swelling from ischemia. The ideal strategy is to both replace OCC after shock (the gold standard), and decompress the microcirculation using impermeant-induced passive water transfer, thus optimizing oxygen transfer to the mitochondria. The perfusion efficiency gained with PEG-20k based crystalloids may dramatically reduce the amount of blood products needed and expand these scarce resources.
The ability of low volume crystalloid resuscitation with 10% PEG-20k to maintain a level of oxygen delivery sufficient to allow 24-hour survival without further replacement of hemoglobin suggests that this solution could serve as a partial blood substitute. Administration of this solution in shocked patients would be particularly useful in austere pre-hospital environments where blood products are unavailable. Perhaps these solutions are also useful in conditions of low blood supply (mass casualty) or simply to augment oxygen delivery of traditional oxygen carriers such as WB or blood products. In fact, preliminary studies suggest PEG-20k added to WB at the time of resuscitation rescues the failure of the blood alone to resuscitate and survive severely shocked swine (unpublished data). Although PEG-20k-based crystalloids alone were able to support recovery from lethal shock up to 24 hours after resuscitation without any additional use of oxygen carriers in the current study, the limits of this effect remain unknown. However, a 24-hour survival period associated with normalization of metabolic and central cardiovascular outcomes after resuscitation from lethal shock would be an enormous advancement in pre-hospital care, medical transport, and early in-hospital resuscitation of the trauma patient.
PEG-20k is rapidly cleared by the kidneys resulting in a 2-hour half-life.23,40 Therefore, the sustained hemodynamic and metabolic effects observed at 24-hours demonstrates the paramount importance of early reversal or prevention of cellular swelling and local microcirculatory failure in achieving effective resuscitation outcomes in severe HS. It seems likely that PEG-20k, through its unique abilities to restore capillary blood flow and oxygen transport after shock, interrupts a vicious cycle responsible for severe and lethal resuscitation injury. Specifically, reversal of cell swelling and early restoration of capillary blood flow sufficiently restores oxygen transfer to local mitochondria such that not only are the bioenergetic requirements of local tissues met, but also the accumulated oxygen debt is repaid. With presumably normalized cell ATP levels to fuel energy dependent cell volume control, the tissues maintain capillary flow after shock, even in the face of a 50% deficit in total OCC. For the first time, these data have unmasked the incredible reserve in oxygen delivery that exists in the system by simply increasing the oxygen transfer efficiency of the microcirculation. If the capillaries can be kept open for exchange, oxygen delivery is adequate even when the OCC is cut by half after severe blood loss. On the other hand, the limited effectiveness with WB and Hextend resuscitation is due to the inability of these components to reproduce the PEG-20k related effect of “fluid repatriation,” which allows utilization of water trapped inside the cells to adequately expand the intravascular space and decrease capillary resistance to flow. The dissatisfactory volume expansion and persistent microcirculatory collapse with WB and Hextend may explain their failure to achieve the same outcomes observed with PEG-20k in our model.
This study only used male pigs for technical reasons. However, the importance of reporting results in both sexes is not overlooked. Female patient groups have significantly better outcomes in shock and trauma compared to males. This effect is not female related per se but rather related to the hormonal milieu of premenopausal and proestrus females.41 These effects are due to estrogen hormone signaling and can be reproduced in males with estrogen and other steroidal signaling approaches.41–43 Female animals used in studies are almost always excluded when they are in estrous so using female pigs in this study may not have introduced an estrogen variable. The large biological effect observed with PEG-20k resuscitation in shock also makes it more difficult that such external variables related to sex may significantly reduce the treatment effect or alter the conclusions of the study. Therefore, it is reasonable to suggest that this study, which only used a small population of male pigs, would not have had different results and conclusions if the same number in the groups were equally distributed between male and female subjects. Upcoming clinical trials for restoring tissue perfusion will obviously require both sexes of patients.
Lastly, the known coagulopathic effects of other polymers like Hextend16 and the effective volume expansion effects of PEG-20k to produce a dilutional coagulopathy has necessitated a comprehensive analysis of the effects of PEG-20k on coagulation and platelet function. Previous ex-vivo analysis of PEG-20k on coagulation and platelet function of blood from healthy volunteers and from severely injured trauma patients during early admission44,45 and in vivo analysis in the same porcine model of HS40 showed only a mild and reversible coagulopathy in the first 2 hours after IV resuscitation. The mechanisms involve a nonspecific functional thrombasthenia and minor effect on Factor XIII-induced fibrin cross linking.40,44,45
The main limitation of this study is related to the experimental design using a controlled hemorrhage model. Although the controlled hemorrhage nature of our model allows accurate scientific comparisons of the mechanistic targets of treatment, there are limitations in clinical translatability when bleeding cannot be controlled at resuscitation. We also did not evaluate other blood components or different plasma: platelet: RBCs ratios that are currently utilized in clinical practice. Instead, we chose to use 2 control solutions that are used in prehospital resuscitation in the military (Hextend) and in the civilian field (WB). Finally, this study only used male pigs but no significant differences in the results of PEG-20k resuscitation impacting our conclusions is expected in female pigs, as discussed above.
In conclusion, LVR with PEG-20k based crystalloid solutions resulted in 100% survival for 24 hours in a lethal HS model, compared to either the same volume of Hextend or WB where most animals survived for about 2 hours. Resuscitation with PEG-20k was associated with return to baseline of plasma lactate, arterial blood pressure, and plasma volume, and capillary perfusion. Survival with PEG-20k was always associated with normal neurological function 24 hours after recovery. This novel crystalloid resuscitation solution may be superior for prehospital use in shocked patients, and it may be efficacious as a partial blood substitute or in combination with blood and blood products. Finally, these data show that WB per se may not be the gold standard for resuscitation and demonstrate that a major mechanism of injury in shock is loss of capillary oxygen transfer due to metabolic cell swelling. The use of blood or other oxygen-carriers in shock without first addressing this mechanism is suboptimal because of inadequate delivery of oxygen to the mitochondria. Based on this study, enhancing both OCC and efficient transit through the microcirculation is the ideal resuscitation strategy.
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