Luciana N. Torres

Evaluation of resuscitation fluids on endothelial glycocalyx, venular blood flow, and coagulation function after hemorrhagic shock in rats.

BACKGROUND Endothelial glycocalyx (EG) plays an essential role in endothelium integrity and may be compromised by hemorrhagic shock. The effects of currently available resuscitation fluids such as Hextend (HEX) or lactated Ringer’s solution (LR) on vascular function and coagulation are not well understood. The aim of the present study was to compare the effects of fresh frozen plasma (FFP) with HEX or LR in their ability to repair EG structure, promote volume expansion, increase blood flow, and prevent coagulopathy. METHODS A total of 121 microvessels from cremaster muscle were studied in 32 anesthetized instrumented rats. After baseline systemic and microvascular measurements, 40% hemorrhage followed by resuscitation was performed, and measurements were repeated. Coagulation was evaluated using ROTEM to assay clot formation time, clotting time, firmness, strength, and lysis. Velocity and “platelet component” of strength were calculated. Fluorescein isothiocyanate or Texas Red bound to Dextrans was injected to estimate EG thickness in vivo. RESULTS Respiratory rate, blood pH, base excess, and lactate returned to near-baseline levels in all treatments. Hemodilution caused by LR and HEX decreased firmness, prolonged clotting time, and lowered platelet counts. EG thickness in HEX- and LR-treated rats was 50% lower, and plasma syndecan 1 was 50% higher than sham and FFP groups. Blood flow and shear rate were restored in the HEX group. Resuscitation with FFP improved coagulation and blood flow. CONCLUSION Our findings support the concept of cardiovascular and microvascular stabilization by infused FFP, in which the increase in microvascular perfusion associated with restored EG is essential for an optimal resuscitation strategy.

Resonance Raman spectroscopy: a new technology for tissue oxygenation monitoring.

Objective:To evaluate resonance Raman spectroscopy for the detection of changes in sublingual mucosal hemoglobin oxygen saturation (Smo2) in response to hemorrhage and resuscitation, and to compare Smo2 with other indicators of tissue oxygenation including central venous oxygen saturation (Scvo2), lactate, base excess, and shed blood volume. Design:Prospective single group pilot study. Setting:University laboratory. Subjects:Five Sprague-Dawley rats. Interventions:Animals were anesthetized and instrumented for measurement of arterial and central venous blood gases. Raman spectroscopy was performed using a krypton ion laser providing excitation at 406.7 nm (5 mW). A 1-mm2 region of the sublingual tongue surface was chosen for investigation. Animals were subjected to stepwise hemorrhage until approximately 50% of the blood volume was removed. At each hemorrhage and resuscitation interval, Raman spectroscopy was performed and corresponding arterial and central venous blood gas and lactate measurements were made. Smo2 was calculated as the ratio of the oxygenated heme spectral peak height to the sum of the oxy- and deoxyhemoglobin spectral peak heights. Raman spectroscopy-derived Smo2 measurements were compared with Scvo2 as well as with other indicators of oxygenation. Measurements and Main Results:The mean difference between Smo2 and Scvo2 for all paired measurements was 5.8 ± 11.7 absolute saturation points. Smo2 was significantly (p < .0001) correlated with Scvo2 (r = .80), lactate (r = −.78), base excess (r = .80), and shed blood volume (r = −.75). Smo2 and Scvo2 showed similar levels of precision for predicting elevated lactate and base deficit. Conclusions:These studies demonstrate the ability of Raman spectroscopy to noninvasively track microvascular hemoglobin oxygenation in tissue and favorably correlate with other important indicators of tissue oxygenation such as Scvo2, lactate, base deficit, and shed blood volume. The technique shows promise as a method to noninvasively monitor tissue oxygenation.

Early physiologic responses to hemorrhagic hypotension

The identification of early indicators of hemorrhagic hypotension (HH) severity may support early therapeutic approaches and bring insights into possible mechanistic implications. However, few systematic investigations of physiologic variables during early stages of hemorrhage are available. We hypothesized that, in certain subjects, early physiologic responses to blood loss are associated with the ability to survive hemorrhage levels that are lethal to subjects that do not present the same responses. Therefore, we examine the relevance of specific systemic changes during and after the bleeding phase of HH. Stepwise hemorrhage, representing prehospital situations, was performed in 44 rats, and measurements were made after each step. Heart and respiratory rates, arterial and venous blood pressures, gases, acid-base status, glucose, lactate, electrolytes, hemoglobin, O(2) saturation, tidal volume, and minute volume were measured before, during, and after bleeding 40% of the total blood volume. Fifty percent of rats survived 100 min (survivors, S) or longer; others were considered nonsurvivors (NS). Our findings were as follows: (1) S and NS subjected to a similar hemorrhage challenge showed significantly different responses during nonlethal levels of bleeding; (2) survivors showed higher blood pressure and ventilation than NS; (3) although pH was lower in NS at later stages, changes in bicarbonate and base excess occurred already during the hemorrhage phase and were higher in NS; and (4) plasma K(+) levels and glucose extraction were higher in NS. We conclude that cardiorespiratory and metabolic responses, essential for the survival at HH, can differentiate between S and NS even before a lethal bleeding was reached.

Systemic and Microvascular Effects of Resuscitation with Blood Products After Severe Hemorrhage in Rats

BACKGROUND Severe hemorrhage is associated with the disruption of the endothelial glycocalyx (EG), a key component of the endothelium. The effects of blood components on the EG are unknown. The present study furthers our investigations into the effects of resuscitation with blood products on the skeletal muscle microcirculation of hemorrhaged rats, focusing on packed red blood cells (PRBCs) or fresh whole blood (FWB). METHODS Rats were bled 40% of total blood volume and resuscitated with 1:1 PRBC/lactated Ringer’s solution (LR), 1:1 washed PRBC (wPRBC)/LR, FWB or LR only. Sham animals were subjected to all procedures except hemorrhage and resuscitation. EG thickness, blood flow, and microvascular permeability were studied using intravital microscopy. Hemodynamics and coagulation tests (rotational thromboelastometry) were performed. RESULTS After severe hemorrhage, EG and permeability were restored to sham levels in the PRBC/LR and FWB groups, but not in the wPRBC/LR or LR groups. Clotting time was longer and clot elasticity and firmness were reduced in wPRBC/LR and LR, but not in FWB or PRBC/LR groups when compared with sham. CONCLUSION Resuscitation with FWB or PRBC/LR was superior in reversing coagulopathy, restoring EG and permeability changes following hemorrhage, compared with wPRBC/LR or LR alone. As wPRBC/LR did not improve EG and permeability, these data suggest that the removal of residual plasma protein from wPRBC or resuscitation with a protein-free solution (LR) is not able to improve microcirculation and coagulation functions in this severe hemorrhage model.

Plasma syndecan-1 and heparan sulfate correlate with microvascular glycocalyx degradation in hemorrhaged rats after different resuscitation fluids

The endothelial glycocalyx plays an essential role in many physiological functions and is damaged after hemorrhage. Fluid resuscitation may further change the glycocalyx after an initial hemorrhage-induced degradation. Plasma levels of syndecan-1 and heparan sulfate have been used as indirect markers for glycocalyx degradation, but the extent to which these measures are representative of the events in the microcirculation is unknown. Using hemorrhage and a wide range of resuscitation fluids, we studied quantitatively the relationship between plasma biomarkers and changes in microvascular parameters, including glycocalyx thickness. Rats were bled 40% of total blood volume and resuscitated with seven different fluids (fresh whole blood, blood products, and crystalloids). Intravital microscopy was used to estimate glycocalyx thickness in >270 postcapillary venules from 58 cremaster preparations in 9 animal groups; other microvascular parameters were measured using noninvasive techniques. Systemic physiological parameters and blood chemistry were simultaneously collected. Changes in glycocalyx thickness were negatively correlated with changes in plasma levels of syndecan-1 (r = -0.937) and heparan sulfate (r = -0.864). Changes in microvascular permeability were positively correlated with changes in both plasma biomarkers (r = 0.8, P < 0.05). Syndecan-1 and heparan sulfate were also positively correlated (r = 0.7, P < 0.05). Except for diameter and permeability, changes in local microcirculatory parameters (red blood cell velocity, blood flow, and wall shear rate) did not correlate with plasma biomarkers or glycocalyx thickness changes. This work provides a quantitative framework supporting plasma syndecan-1 and heparan sulfate as valuable clinical biomarkers of glycocalyx shedding that may be useful in guiding resuscitation strategies following hemorrhage.

Refrigerated platelets stored in whole blood up to 5 days adhere to thrombi formed during hemorrhagic hypotension in rats.

Essentials In vivo function of platelets stored at various conditions was studied in normo‐ and hypotension. Refrigerated platelets stored up to 5 days performed as well as those stored at room temperature. Platelet adhesion and thrombus formation were higher in ruptured vessels of hemorrhaged animals. In vivo data suggest that refrigerated platelets are hemostatically effective during hypotension.

Effects of perfluorocarbon emulsions on microvascular blood flow and oxygen transport in a model of severe arterial gas embolism.

BACKGROUND Arterial gas embolism (AGE) is a clinical problem that occurs directly in cardiopulmonary bypass machines in open-heart surgeries, or indirectly (through cardiac or pulmonary right to left shunts) in dive accidents, resulting in serious morbidity and even death. Perfluorocarbon (PFC) emulsions have been used for the treatment of AGE in an animal model. We hypothesized that PFC emulsions enhance microvascular blood flow, speed bubble resolution, and oxygenation in AGE compared with saline in a model of cremaster muscle from anesthetized rats. MATERIALS AND METHODS AGE was induced by direct air injection into the femoral artery ipsilateral to the studied cremaster muscle. Microhemodynamics, microvascular, and tissue oxygenation were determined before and after treatment with two different commercial PFC emulsions (C10F20, Oxycyte; Oxygen Biotherapeutics, Inc and C10F18, PHER-O2; Sanguine Corporation, Inc) compared with saline in real time using brightfield and phosphorescence microscopy. RESULTS Blood pressure and heart rate remained unchanged. Systemic PO2, oxygen (O2) content, and glucose were higher in PFC groups, whereas hematocrit dropped in all groups. Arteriolar blood flow went up 85% and 80% of baseline after C10F20 and C10F18 treatments, respectively, versus 11% after saline treatment. Arteriolar and tissue PO2, and O2 delivery were higher in PFC groups compared with the control group. There was an increase in arteriolar blood flow, reduction in diffusional resistance of O2 in the plasma, and improved tissue oxygenation. CONCLUSIONS Administration of PFC emulsions in AGE is superior to saline primarily because of surfactant properties along with air bubble reabsorption.

In vivo microvascular mosaics show air embolism reduction after perfluorocarbon emulsion treatment.

Massive arteriolar gas embolism (AGE) has never been evaluated in vivo using intravital microscopy and previous perfluorocarbon (PFC) emulsions were only effective in AGE when administered before AGE. We implemented a new system for quantitative studies of massive AGE using brightfield microscopy and tested a treatment with a third-generation PFC emulsion after massive AGE. We studied bubble dynamics in cremaster muscles from anesthetized rats after AGE was induced by direct air injection into the femoral artery ipsilateral to the studied muscle. Using a motorized microscope stage and a color camera, in vivo microvascular mosaics were produced on-line from over 2000 digital images to evaluate multiple networks in order to investigate the distribution, lodging, breaking, reduction and moving of 105 air bubbles in microvessels. Thirty minutes after PFC treatment, there was a reduction of 80% in bubble volume while untreated and saline-treated rats showed significantly smaller decreases of 33% and 40%, respectively (p<0.05). Air bubbles also dissolved into a larger number of smaller bubbles after PFC treatment. The proposed methodology may prove useful for rapid in vivo data acquisition from large networks. Since large air bubbles broke-up, decreased in length and volume, and moved toward smaller microvessels, the study provides quantitative data to support a mechanism by which PFC may improve tissue blood flow following massive AGE. The findings suggest that this new generation of PFC emulsions administered after severe AGE may reach compromised microvascular networks and provide help to alleviate microvascular obstruction by increasing air bubble reabsorption.

1588: IMPACT OF NORMAL SALINE RESUSCITATION ON ENDOTHELIAL FUNCTION AFTER HEMORRHAGE IN RATS

Learning Objectives: Successful resuscitation requires mitigation of endothelial injury and restoration of microcirculatory function. With an underlying link between endothelial glycocalyx (EG) integrity and vascular homeostasis, EG protection may be an important therapeutic target. Normal saline (NS) is normally used for resuscitation from hemorrhage and other conditions in critically ill patients, but its use has become controversial in recent years. This study compared effects of NS and lactated Ringer’s (LR) on their ability to maintain EG, barrier function and other indices of microvascular function in vivo in 180 venules from cremaster muscle in 36 anesthetized hemorrhaged rats. Methods: Rat fresh frozen plasma (FFP) was included as a positive control. Rats were bled 40% of blood volume and resuscitated with 45 ml/kg of NS or LR, or 15ml/kg of FFP. Mean arterial pressure (MAP), heart rate, blood gases, biochemistry and viscosity were also monitored. Results: Shock compromised acid-base balance, but resuscitation normalized pH, lactate and base deficit in all groups. MAP was 25% lower after resuscitation with all fluids (vs. baseline, 101 ± 5mmHg). Hematocrit and viscosity decreased in all groups, but plasma protein was further reduced in NS and LR (vs. FFP). As expected, NS significantly increased plasma Cland Na+ vs. LR. Shock caused significant EG shedding and no fluid restored thickness to baseline (0.57 ± 0.02 μm), except for FFP. FFP infusion stabilized permeability and WBC rolling/adhesion vs. baseline. NS decreased blood flow and shear, and increased permeability and WBC rolling/adhesion. In contrast, LR had lesser effects on increased permeability and WBC rolling. Conclusions: Crystalloids, particularly NS infusion, could not restore EG components after shock leading to a deleterious impact on microvascular functions. Our findings provide physiological basis to re-evaluate NS as fluid resuscitation therapy in critical care and highlight the potential harmful effects of NS compared to balanced crystalloids and FFP on endothelial and microvascular stabilization after shock.