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Departments of *Medical Pathology, Anesthesiology and Pain Medicine, and Surgery, University of California, Davis School of Medicine, Davis, California; and Department of Clinical Studies, New Bolton Center, University of Pennsylvania School of Veterinary Medicine, Kennett Square, Pennsylvania
Address correspondence and reprint requests to Professor Anthony T. W. Cheung, Department of Medical Pathology, Research-III Building (Suite 3400), UC Davis Medical Center, 4645 Second Ave., Sacramento, CA 95817. Address e-mail to atcheung{at}ucdavis.edu
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Abstract |
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40% blood loss) reduced mean arterial pressure to 50 mm Hg and caused significant (P < 0.01) decreases in hematocrit, total hemoglobin, mean pulmonary arterial pressure, cardiac output, and oxygen delivery and significant (P < 0.01) increases in heart rate, systemic vascular resistance, and lactic acidosis. Significant (P < 0.01) changes in conjunctival microvascular variables also occurred, including a 19% decrease in venular diameter and 79% increase in average blood flow velocity. Shed blood resuscitation returned microvascular, systemic function, hemodynamic, and oxygenation variables to prehemorrhagic baseline values. In contrast, Hb-200 failed to restore hematocrit, total hemoglobin, cardiac output, oxygen delivery index, and systemic venous resistance to baseline, but it restored other systemic functions and all hemodynamic and microvascular changes. In addition, Hb-200 resuscitation in hypovolemic dogs (40% blood loss) did not cause extreme hemodilution or fatal outcome. This study confirms that real-time (in vivo) microvascular studies, which were conducted only in small rodent models in the past, can be performed simultaneously with systemic function, hemodynamic, and oxygenation studies in a large animal model for relevant data correlation. IMPLICATIONS: This is the first time that changes in the blood circulation have been studied, quantified, and correlated with systemic function, hemodynamic, and oxygenation changes in shock and during shock treatment in a large animal model. This study was performed by a new technology developed in-house to noninvasively and quantitatively study blood vessels in real time.
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Introduction |
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The development of these products is a formidable task that has been hindered in part by the fact that the effects of these blood substitutes on the microcirculation have not been fully appreciated, and the products developed have not been engineered so that their physical properties are congruent with those of the microcirculation (1,7). Restoration of systemic function, biochemistry, and oxygenation variables by HBOC resuscitation after acute blood loss has been a goal for pharmaceutical and biotechnological companies and has been studied in a variety of animal models. However, there has been little or no emphasis on the behavior of the HBOC in the microcirculation, the organ system in which blood performs its tissue-level physiologic function (7). Most blood substitutes, including all HBOC, are basically oxygen-carrying plasma expanders and, as such, can cause extensive hemodilution under severe hypovolemic conditions; the hematocrit (Hct) can be reduced to less than the transfusion trigger during resuscitation, resulting in a paradoxical effect of producing or exacerbating vasoconstriction (8,9) instead of alleviating or reversing hypovolemic complications—an outcome that is opposite to what is needed and jeopardizes the goal of resuscitation. In vitro experiments have provided evidence that the hemoglobin (Hb) molecule itself can interfere with physiologic mechanisms that control vasomotor tone. It is thought that scavenging of the endothelium-derived relaxing factor, nitric oxide, is the chief mechanism by which free Hb can elicit vasoconstriction in both arterial and venous sites, but release of endothelin-1 and interactions with adrenoreceptors and inositol triphosphate pathways can also be involved in vasoconstriction (10,11). To fully evaluate the functionality, efficacy, and toxicity of HBOC, detailed studies on their effects on the microcirculation are warranted.
To adequately assess inherent complications arising from HBOC resuscitation, we used a canine hypovolemia model to study the real-time effects of hemoglobin glutamer-200 (bovine) (Hb-200) (Biopure Corp., Cambridge, MA) on the postresuscitation microcirculation. This study is the first of its kind to study the in vivo microvascular activities during hypovolemic shock resuscitation and to correlate these results with systemic function, hemodynamic, and oxygenation characteristics in a large animal model (12–14). Hb-200 was used as the resuscitation fluid because this compound has been approved by the Food and Drug Administration for canine use (15). Because of the similarity of Hb-200 to Hb-201 (Hemopure®; Biopure Corp.), which has been approved by the Food and Drug Administration for human phase III clinical trials, this study has a direct technology transfer orientation and translational significance.
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Methods |
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In brief, each dog was premedicated with IM oxymorphone 0.02 mg/kg and atropine 0.02 mg/kg, followed by percutaneous catheterization of the cephalic vein for continuous infusion of lactated Ringer’s solution at a rate of 10 mL · kg-1 · h-1 throughout the preparation and instrumentation period. The dog was anesthetized with IV propofol 2–4 mg/kg and diazepam 0.5 mg/kg, subsequently intubated, and mechanically ventilated with a small-animal anesthesia machine (Frazer Harlake, Orchard Park, NY) operating with an IsotecTM vaporizer (Ohmeda, Milwaukee, WI). During animal preparation and instrumentation, anesthesia was maintained with isoflurane in 100% oxygen (end-tidal concentration of isoflurane 0.8%–1.2%) and an infusion of fentanyl at a rate of 0.7 µg · kg-1 · min-1 after an initial bolus of 10 µg/kg fentanyl. The dog was then splenectomized and instrumented for experimentation in dorsal recumbency by following a protocol established in our laboratory (12–14). The spleen of the dog has a unique capability to sequester and store red blood cells. These cells are normally returned to the circulation under some conditions of sympathetic nervous system stimulation (including hypovolemic shock). Therefore, the splenectomy was performed to prevent this complication. Instrumentation included insertion of catheters into the dog’s cephalic vein, lateral saphenous vein, and femoral arteries for drug and fluid administration, blood withdrawal, and determinations of systolic arterial pressure (SAP), diastolic arterial pressure (DAP), and mean arterial pressure (MAP), as well as placement of a balloon-tipped pulmonary catheter via the jugular vein for measurements of the cardiac output (CO), pulmonary occlusion pressure (POP), and central venous pressure (CVP). After instrumentation, heart rate (HR), SAP, DAP, MAP, CO, mean pulmonary arterial pressure, POP, and CVP were continuously monitored. Body temperature was maintained between 37°C and 39°C by means of a heating pad and circulating warm air blanket (Bair Hugger® Model 505; Augustine Medical Inc., Eden, MN) placed underneath and on top of the animal, respectively. Hct, Hb, arterial oxygen content (CaO2), venous oxygen content (CvO2), and lactate concentration were analyzed in collected blood samples, and stroke volume index (SVI), oxygen delivery index (DO2I), and oxygen consumption index (
O2I) were calculated as described previously (12–14). Hct was measured by means of capillary tube centrifugation, total and plasma Hb were measured in appropriate samples by using a Nova cooximeter (Nova Biomedical, Waltham, MA), and CaO2 and CvO2 were directly determined in duplicate by using an oxygen-specific electrode (LEXO2CON-K; Hospex Fiberoptics, Chestnut Hill, MA). Arterial lactate concentration was determined in duplicate by using a Sport Lactate Analyzer (Model 1500; YSI, Yellow Springs, OH). Arterial pH and arterial pressures were analyzed with a blood gas analyzer (Model 170; Corning Medical, Medfield, MA). Blood gas values were corrected for body temperature of the animals at the time of the sampling. Cardiac index (CI), SVI, and systemic vascular resistance (SVR) were calculated with standard formulae. Red-cell Hb, if needed, was calculated as the difference between total Hb and plasma Hb. DO2I was calculated as CaO2 x CI, and O2I was calculated as (CaO2 - CvO2) x CI.
The intravital microscopy protocol was adapted from a noninvasive and real-time (in vivo) methodology that was described in detail in a previous report (16). The intravital microscope was stably mounted on a portable stand positioned at the side of the operating table. Videotapes on the conjunctival microcirculation were made in all phases (periods) of the experiments. Under this intravital setting, the conjunctival vessels appeared on-screen as crisp black lines or tubes on a white background (see Fig. 1A–D). On-screen focusing was conducted repeatedly to ensure sharp image display. The videotapes were viewed in their entirety after the experiments. Well-resolved videotape sequences to be analyzed were selected and coded; the coded video sequences were given to investigators at random to analyze for morphometric (diameter and distribution) and dynamic (blood flow velocity) characteristics by using in-house developed imaging software, VASCAN and VASVEL (16,17). The investigator conducting the analysis was blinded from the source of the video sequences and experimental details. All measurements from the same phase of the study in the same animal were averaged (mean ± SD). Data from the same phase of the study in different animals were categorized, averaged, and statistically analyzed. The microvascular variables sought included venular diameter, length of vessels (arterioles and venules), arteriole/venule (A/V) ratio, and blood flow velocity. The computer-assisted data analysis protocol was described in detail in previous articles (16,17).
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85 mL/kg body weight) was withdrawn from the lateral saphenous vein and femoral artery until a MAP of 50 mm Hg was reached. This hemorrhage method of attaining a MAP of 50 mm Hg as a clinical criterion was used to ensure the induction of acute but nonlethal hypovolemia and was normally completed within 20–30 min. Shed blood was collected, anticoagulated (citrate phosphate dextrose), and weighed to determine total blood volume loss on the basis of weight (1 g = 1 mL). Additional small amounts of blood were withdrawn or infused when needed to maintain a MAP of 50 mm Hg for 60 min. At the end of 1 h of sustained moderate hypovolemia (posthemorrhagic period), all measurements and videotape recordings were repeated. Then, each dog was randomly assigned to either the Control or Experimental group. Control animals (n = 4) were resuscitated by reinfusion of autologous shed blood at a rate of 30 mL · kg-1 · h-1, whereas the experimental animals (n = 4) received Hb-200 at a rate of 10 mL · kg-1 · h-1, as recommended by the manufacturer. Transfusion of shed blood or Hb-200 was discontinued once HR and MAP returned to baseline values. All measurements and videotapes were made immediately after the termination of resuscitation (Resuscitation 1 period). The animal was maintained for three additional hours (observation period), after which final measurements and videotapes were made (Resuscitation 2 period). The videotapes on the conjunctival microcirculation were objectively analyzed as described previously. All animals remained anesthetized throughout the procedure and were killed at the end of the Resuscitation 2 period with an overdose of potassium chloride without their regaining consciousness. All results were averaged and reported as mean ± SD. Analysis of variance, Student’s t-tests, and post hoc Bonferroni corrections were used whenever appropriate. A 0.05 significance level was used in this study. P values <0.01 (e.g., P = 0.000045 or P = 5.793 x 10-4) were presented as P < 0.01 for simplicity.
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Results |
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The total amount of blood removed during hemorrhage ranged between 32 and 36 mL/kg in the animals, corresponding to 40% (38%–41%) of the estimated canine circulating blood volume (85 mL/kg body weight). Posthemorrhagic measurements showed that arterial Hct, total Hb, and CaO2 decreased close to the same extent (15%–18%) in all animals. In general, hypovolemia induced by hemorrhaging resulted in significant decreases in Hct, Hb, SAP, DAP, SVI, CO, CI, DO2I, CaO2, and CvO2, as well as in significant increases in HR, SVR, and lactic acidosis. There was no significant change in posthemorrhagic O2I values. The results are summarized in Tables 1 and 2.
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1:2; flow velocity = 0.5 ± 0.3 mm/s; n = 8). After hemorrhage, all eight dogs in both groups showed similar significant (P < 0.01) posthemorrhagic responses: a 19% decrease in vessel diameter (diameter of venules = 33 ± 8 µm), a change of vessel density (A/V ratio = extremely variable), and a 79% increase in blood flow velocity (blood flow velocity = 0.9 ± 0.4 mm/s). Because there were no differences between groups at baseline and posthemorrhage values, data from all the dogs were averaged and are summarized (Table 3).
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Both resuscitations with autologous shed blood and Hb-200 led to significant (P < 0.01) microvascular improvements. Shed blood resuscitation (30 mL · kg-1 · h-1) returned all microvascular variables to baseline prehemorrhagic level (diameter of venules = 39 ± 6 µm; A/V ratio = 1:2; flow velocity = 0.6 ± 0.4 mm/s; P < 0.01). Hb-200 resuscitation (10 mL · kg-1 · h-1) also significantly restored microvascular variables close to prehemorrhagic values (diameter of venules = 38 ± 3 µm; A/V ratio = 1:2; flow velocity = 0.6 ± 0.4 mm/s; P < 0.01). The results are summarized in Table 3. The microvascular improvements in both shed blood and HBOC resuscitation groups were not significantly different immediately and 3 h after resuscitation.
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Discussion |
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40% of the circulating blood volume with a Hct reduction of 15%–18%. It should, therefore, be emphasized that this report describes a resuscitation study on moderate but not severe hypovolemic shock. The goal of this pilot study was to confirm that the microcirculation of the animals can be studied simultaneously and correlated with systemic function, hemodynamic, and oxygenation changes in a large-animal model. Because it was a pilot study, only eight animals (four experimental animals with four controls) were used. With the limited number of animals used in this pilot study, the results obtained are interesting and suggestive but should not be considered conclusive. A follow-up study is planned and will involve a much larger number of animals (n = 20 for each group). In addition, the blood loss will be significantly increased to approach the transfusion trigger (50%–60% loss of circulating blood volume with an Hct reduction of 
40%). Autologous shed blood resuscitation (30 mL/kg) restored all systemic and hemodynamic functions and oxygenation characteristics to baseline prehemorrhagic values. However, Hb-200 resuscitation restored only some of the systemic function and oxygenation characteristics in this study. The discrepancy may be explained by differences in the composition of blood and Hb-200 (e.g., total solid and viscosity) and possibly inadequate resuscitation volume because of the infusion rate recommended by the manufacturer of Hb-200.
Under clinical conditions, volume replacement with autologous shed blood resuscitation is most often guided by measurements of HR and MAP because more detailed hemodynamic data are rarely, if ever, available at the time of resuscitation. By following standard blood transfusion protocols, we transfused shed blood at a rate of 30 mL · kg-1 · h-1, a rate used in previous work in this and other laboratories. To evaluate the effects of Hb-200 under conditions as close as possible to the clinical scenario, we also used a return of HR and MAP to baseline as the end point of volume resuscitation, followed by continuous postresuscitation monitoring. Hb-200 was administered to the hypovolemic dogs at the rate recommended by the manufacturer, warning of excessive circulatory overload in case of rapid fluid administration (10 mL · kg-1 · h-1 corresponding to 1.3 g · kg-1 · h-1 of bovine Hb infusion). It should be noted, however, that both shed blood and Hb-200 resuscitations were completed within the same time frame to obtain measurements at the same time intervals in both groups despite the different infusion rates used. As expected, shed blood resuscitation resulted in the restoration of all systemic function, hemodynamic, and oxygenation variables to baseline prehemorrhagic values. In contrast to shed blood resuscitation, Hb-200 resuscitation did not restore most oxygenation characteristics and CO to prehemorrhagic values. Thus, animals resuscitated with Hb-200 were systemically underresuscitated, despite the restoration of HR and MAP to prehemorrhagic values. It should be noted, however, that CO could remain at a low level as a result of the increase in total peripheral vascular resistance caused by Hb-200 and not primarily because of volume underloading, because CVP and POP returned to prehemorrhage baseline values.
Regardless of the significantly low oxygenation characteristics in the Hb-200 Resuscitation group, we did not find any evidence for persistence of global tissue hypoxia in this group when compared with the Shed Blood Resuscitation group. Arterial lactate values returned in both resuscitation groups to or near baseline values after resuscitations. To maintain oxygen consumption, tissues in HBOC-Resuscitated dogs markedly increased oxygen extraction from arterial blood; this was evident from the significantly lower mixed-venous oxygenation measurements in this study. One might, therefore, speculate either that blood flow at the tissue level was less compromised than increased SVR indicated or that despite increased SVR, the diffusive component of oxygen transport and the capillary oxygen transport to the cells were improved after Hb-200 resuscitation.
As expected, shed blood resuscitation resulted in the restoration of all microvascular characteristics to baseline values. Hb-200 resuscitation also restored all microvascular characteristics to baseline values. In addition, this study confirms that Hb-200 does not cause extreme hemodilution or induce any toxic or lethal effect when used as a resuscitation drug in moderate hypovolemia over a three-hour observation period. On the basis of this study, we plan to extend our investigation to evaluate the effect of Hb-200 when blood loss is at 50%–60% with an Hct reduction of 40% (close to the transfusion trigger). Even though the effects of extreme hemodilution were not evaluated in this study, it represents the first study of its kind in which real-time microvascular changes have been documented and studied in a large laboratory animal model. In addition, this is also the first time that the effects of HBOC resuscitation on the microcirculation were studied and correlated with systemic function, hemodynamic, and oxygenation changes in the same animal.
The conjunctival microcirculation was chosen as the site to study the microcirculation in vivo because of its easy and noninvasive accessibility. In addition, individual conjunctival vessels were easily recognizable and could be relocated in time-dependent studies for longitudinal reference (see Fig. 1A–D). This study also shows that the computer-assisted intravital microscopy technology may represent the availability of a powerful, noninvasive, objective, and quantitative tool to study the microcirculation in vivo in future hemorrhagic shock and HBOC resuscitation studies.
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Acknowledgments |
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Footnotes |
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