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Effect of Trauma-Hemorrhagic Shock on Red Blood Cell Deformability and Shape

Zaets, Sergey B.*; Berezina, Tamara L.*; Morgan, Claire*; Kamiyama, Mikio*; Spolarics, Zoltan; Xu, Da-Zhong*; Deitch, Edwin A.*; Machiedo, George W.*

Basic Science Aspects
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Previous work in our laboratory has demonstrated a decrease in red blood cell (RBC) deformability in sepsis. This has not been studied following hemorrhagic shock. We tested the hypotheses that hemorrhagic shock, associated with soft tissue trauma, leads to decreased RBC deformability and that this is related to alterations in the resting shape of the RBC. Elongation index (EI), a measure of RBC deformability, was determined over a range of shear stresses from 0.3 to 30 Pa in 26 male rats before and at various times after 90 min of hemorrhagic shock. RBC resting shape was determined by scanning electron microscopy. The data demonstrate that EI decreased significantly at the end of shock (before resuscitation), and remained below normal throughout the 6-h postshock period. Eight of the 26 animals decompensated during shock, requiring return of a portion of the shed blood to maintain a mean arterial pressure of 30–40 mmHg. Four of eight decompensated animals died before the end of the study period, compared with none of the compensated rats. The decompensated rats had significantly lower EI at 0.3 Pa by the end of the shock period (0.050 ± 0.009) than the compensated shock group (0.058 ± 0.006;P < 0.05). RBC shape alterations were first demonstrated at the end of the shock period and persisted throughout the 6-h postshock resuscitation period. These data indicate that trauma and hemorrhagic shock cause RBC shape alterations and a significant decrease in RBC deformability, which becomes manifested during the shock period and persists for at least 6 h postshock. Additionally, a direct relationship appears to exist between the magnitude of the physiologic insult and the degree of RBC damage.

*Departments of Surgery and Anatomy, Cell Biology, and Injury Science, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103-2714

Received 6 Jun 2002;

first review completed 1 Jul 2002; accepted in final form14 Aug 2002

Address reprint requests to Sergey B. Zaets, MD, Department of Surgery, MSB, Room G-507, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103-2714.

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INTRODUCTION

Previous studies (1) using intravital microscopy have demonstrated that in hemorrhagic shock, crenated red blood cells (RBC) are seen within arterioles, venules, and capillaries and that during refractory shock, nearly all red cells become crenated spheres. These observations that shock is associated with the production of echinocytes in vivo represent morphological evidence of the damage to RBC caused by hemorrhage. Of potential clinical importance is the fact that these damaged RBCs may no longer able to pass through the previously negotiable microcirculatory bed, which in turn may lead to decreased microvascular flow and local hypoxia, thereby contributing to the development of multiple organ failure. However, in vivo data concerning changes in RBC deformability during and after hemorrhagic shock are limited and contradictory. For example, Likhovetskaia et al. (2) showed a decrease in RBC deformability during all stages of hemorrhagic shock. In contrast, Matsumoto (3) and Sigiura et al. (4) reported that RBC deformability remained normal during hemorrhagic shock, whereas in septic shock, the deformability significantly decreased. In fact, Soloviev (5) even observed a compensatory increase in RBC deformability after hemorrhagic shock. The fact that these authors used different methods for the determination of RBC deformability, including various filtration techniques and RBC morphometry after centrifugation, makes data interpretation difficult because each of the methods used was either indirect or their results can be influenced by the presence of white blood cells (WBC). One potential solution to this problem is the use of the recently described method of laser-assisted ektacytometry (LORCA) because this method of measuring deformability is specific for RBC and is not affected by the presence of WBC (6). Although LORCA has been used successfully for measuring RBC deformability in septic shock (6), we are not aware of any studies examining RBC deformability in hemorrhagic shock by means of ektacytometry. Consequently, the aim of this study was to investigate RBC deformability after trauma and hemorrhagic shock by means of LORCA and to test the hypotheses that hemorrhagic shock leads to decreased RBC deformability and that changes in RBC deformability are related to alterations in RBC resting shape.

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MATERIALS AND METHODS

Animals preparation and trauma/hemorrhagic shock protocol

Twenty-six male Sprague-Dawley rats underwent blood withdrawal according to a standard trauma/hemorrhagic shock protocol (7) that was approved by the institutional Animal Care and Use Committee. The experiments were performed in adherence to the National Institutes of Health Guidelines on the Use of Laboratory Animals. The rats were anesthetized with sodium pentobarbital (50 mg/kg) injected intraperitoneally, and the right femoral artery was isolated by minimal dissection and was aseptically cannulated with polyethylene (PE-50) tubing containing 0.1 mL of heparinized saline. The catheter was connected in-line to a blood pressure recorder and polygraph (model 79E Data Recorder; Grass, Quincy, MA) to allow continuous blood pressure monitoring. Using aseptic technique, the right external jugular vein was cannulated with a 50-gauge silicone catheter containing 0.1 mL of heparinized saline. After a traumatic injury (laparotomy) was created, the abdomen was subsequently closed in two layers using a running 4.0 silk suture. To induce shock, blood was withdrawn from the jugular vein into a syringe containing 10 units of heparin in 0.3 mL of 0.9% normal saline. The mean arterial blood pressure (MAP) was reduced to 30 mmHg and was maintained at this level for 90 min by the careful withdrawal or reinfusion of shed blood (kept at 37°C) as needed. Blood samples (15 μL) were withdrawn for the analysis prior to hemorrhagic shock after 90 min of shock, but before the resuscitation with shed blood, and hourly for 6 additional hours after resuscitation.

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Ektacytometry

RBC deformability was determined at various shear stresses by laser diffraction analysis, using an ektacytometer (LORCA; RR Mechatronics, Hoorn, The Netherlands). The instrument consists of a laser diode, thermostated bob-cup measuring system, stepper motor, and a video camera attached to a microcomputer. The microcomputer controls the speed of the stepper motor, and thus the rotational speed of the cup, to generate various shear stresses in the dilute blood sample. For the study, 15 μL of each sample was suspended in 3 mL of a highly viscous solution of 360,000 molecular weight polyvinylpyrrolidone in an isotonic phosphate buffer base (pH 7.4), and gently rotated at room temperature for 10 min to insure complete oxygenation of the hemoglobin. The shear stresses were applied to the RBC samples in a concentric-cylinder system made of glass, with a gap of 0.3 mm between the cylinders, and rotated to develop shear stresses from 0.3 to 29.9 Pa. The laser beam projected through the sample and the diffraction pattern produced by RBC was analyzed by the microcomputer. RBC elongation index (EI) was calculated by dividing the difference between the long and short axes by the sum of the two axes:A − B/A + B.

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Scanning electron microscopy

Scanning electron microscopic studies of RBC were performed by means of a AMRAY 1200B electron microscope. The blood samples were fixed in phosphate-buffered (pH 7.2–7.4) 2.5% glutaraldehyde for 1 h, washed two times in 0.1 M phosphate buffer (pH 7.2–7.4), and mounted on poly-l-lysine-coated glass slides. The glass slides were kept in a moist atmosphere for 1 h, washed in phosphate buffer, postfixed in 1% osmium tetroxide for 1 h, rinsed in distillated water, and dehydrated in graded ethanol (50%, 70%, 90%, and 100%). After critical point drying with liquid CO2 in a vacuum apparatus and covering with a gold-palladium layer, the samples underwent scanning electron microscopic analysis.

The different cell shapes were identified using Bessis' classification (8). The percentage of discocytes, echinocytes, spheroechinocytes, stomatocytes, spherostomatocytes, and spherocytes was evaluated by counting 500 to 1500 cells in randomly chosen fields. RBC manifesting echinocyte and stomatocyte shapes are capable of returning to the discocyte shape under certain conditions (for example, by bathing in fresh normal plasma). Thus, these RBC shape changes are considered as potentially reversible transformations. In contrast, RBC assuming spheroechinocyte, spherostomatocyte, spherocyte, ovalocytes, and degenerated shapes are irreversibly changed cells.

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Statistical analysis

Data were analyzed using SPSS 9.0 for Windows (SPSS Inc., Chicago, IL), and ARE presented as the mean ± sd. Mean values were compared using analysis of variance (ANOVA) followed by Tukey's test. The level of statistical significance was set at P < 0.05.

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RESULTS

RBC deformability as measured by LORCA ektacytometry (EI) significantly decreased after 90 min of shock (before resuscitation) at a shear stress of 0.30, 0.95, and 29.99 Pa, compared with preshock values (Table 1). Following resuscitation, EI measured at low shear stress remained significantly below normal over the 6-h postshock period. However, over the same period of time, the values of EI at high shear stresses (>1.69 Pa) returned to normal and did not differ significantly from preshock values. EI 1, 3, and 6 h postshock did not significantly differ from its values at the end of shock.

Table 1

Table 1

Eighteen rats remained compensated throughout shock and needed periodic withdrawal of small portions of additional blood during the shock period to maintain the MAP at 30 mmHg. Eight animals decompensated during the 90-min shock period and needed reinfusion of shed blood to maintain a MAP of 30 mmHg. Four of eight decompensated rats died before the end of study, whereas all compensated animals survived the duration of the experiment. The volume of blood withdrawal was significantly lower in decompensated rats compared with that of compensated animals (19.6 ± 2.4 mL/kg and 26.0 ± 4.1 mL/kg, P < 0.01). Additionally, the EI at the end of the 90-min shock period was significantly lower in the decompensated than the compensated rats when tested at a shear stress of 0.30 Pa (0.050 ± 0.009 vs. 0.058 ± 0.006, P < 0.05). It should be mentioned that preshock values of rheological parameters did not differ between the groups of animals.

Scanning electron microscopy performed before the induction of hemorrhagic shock showed the presence of mainly normal biconcave RBC, but also a small number of abnormal cells, mainly reversibly changed erythrocytes (Fig. 1). Pronounced RBC shape alterations were noticed at the end of the shock period, but before resuscitation. At this time point, the number of abnormal RBC (reversibly and irreversibly changed erythrocytes) was significantly increased, whereas the number of normal discocytes had decreased (Table 2). The most dramatic increase was observed in the percentage of reversibly changed forms. They were mostly represented by echinocytes (Fig. 2): echinocytes I, irregularly contoured discs; echinocytes II, flat cells with spicules, and echinocytes III, ovoid cells with multiple spicules. Occasionally, stomatocytes I–III (cells with more or less profound cup shape) could be seen. Irreversibly changed RBC were represented by spheroechinocytes (spheres with short spicules), spherostomatocytes (spheres with depressions), spherocytes (spherical cells), and degenerated forms (cells with ambiguous shape and their fragments). These morphological changes persisted throughout the 6-h postshock period (Fig. 3). As the postshock period progressed, there was a tendency toward a further increase in the percentage of irreversibly changed RBC (Table 2). However, the difference did not achieve statistical significance. The above-mentioned types of irreversibly changed RBC could be seen during the postshock period. Reversibly changed cells, which dominated among abnormal RBC, were still mostly represented by echinocytes. Linear regression showed a tight correlation between the percentage of abnormally shaped RBC (reversibly and irreversibly changed cells) and the elongation index (r = −0.84, P < 0.0001;Fig. 4).

Table 2

Table 2

Fig. 1

Fig. 1

Fig. 2

Fig. 2

Fig. 3

Fig. 3

Fig. 4

Fig. 4

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DISCUSSION

During different types of shock, RBC are subjected to various pathologic influences. Oxygen free radicals, toxins, depletion of ATP stores, alterations of intracellular ionic composition, and compliment activation are all factors decreasing RBC deformability, as well as leading to alteration of their shape. The primary targets are membrane skeletal proteins and lipid bilayer. It has been demonstrated that hypoxia causes a decrease in RBC deformability (9). RBC after hypoxic incubation are more spherical with less excess surface area (10). There is also a relationship between the serum osmolarity and RBC deformability and shape. Hyperosmolarity leads to RBC shrinkage, whereas hypoosmolarity causes RBC swelling. Both processes significantly decrease RBC deformability (11,12).

Shock of any etiology can be regarded as a crisis of the microcirculation. A number of intravital video microscopy studies performed mostly in septic shock models clearly demonstrate capillary occlusion by poorly deformable RBC (13), which might lead to impaired oxygen delivery to organs and tissues. For example, Bateman et al. (13) showed that sepsis-induced decrease in RBC deformability contributes to decreased functional capillary density in skeletal muscle. Preferential entrapment of less deformable RBC has been observed in the microcirculation of lungs, liver, spleen, and bones, causing an increase in resistance and a decrease in blood flow in these organs (14). Furthermore, other studies in septic shock have documented that impaired RBC deformability correlates with decreased transcutaneous oxygen pressure (15). Lastly, chemically induced RBC shape and deformability changes have been shown to decrease RBC flow velocity in capillaries (16) and impair oxygen transport experimentally (17), a finding that was confirmed by clinical studies (18) as well as by means of mathematical modeling (19). Even relatively small decrease in RBC deformability may have clinical impact and may result in microcirculatory disorders. According to Cicco and Pirelli (18), a 10% decrease in RBC deformability in patients who are hypertensive is accompanied by significant impairment of cellular oxygen delivery and tissue oxygenation. Six percent improvement in RBC deformability observed in the same category of patients after treatment resulted in concomitant improvement of the above-mentioned physiological parameters (20).

There are consistent data showing significant RBC deformability and shape abnormalities in septic shock and sepsis (21,22), burn shock and burns (23,24), cardiogenic shock (25), as well as during blood storage (26). The effects of hemorrhagic shock on RBC morphological and rheological properties are much less widely studied, and the results that have been obtained are contradictory (2–5). We believe that these contradictory observations are related to the confounding effects of the various methodologies used in these studies. LORCA is considered to be one of the most reliable techniques to measure RBC deformability. It has been compared with other methods of measuring RBC deformability. Our previous work has shown that Reid filtration technique results correlate with LORCA data (27). However, filtration methods require removal of WBC, which can significantly influence the results. Different washing procedures may not be sufficiently effective to completely remove WBC from RBC suspensions (28). In addition, the Reid filtration method requires a larger volume of blood for each study, and cannot be used for repeated measurements. Computerized cell transit analyzer (CTA) is a new generation cell filtration system, which is less sensitive to WBC-related artifacts. However, this method appeared to be not sensitive enough to detect those sepsis-induced RBC deformability changes that were identified by LORCA (6). Because these methodological concerns can be obviated by the use of LORCA, we used this system to evaluate the effects of trauma-hemorrhage on RBC deformability. It should be also mentioned that LORCA indices correlate with physiologic effects by other investigators. The decrease in RBC deformability (evaluated with LORCA EI) seen in patients with arterial hypertension was associated with a decrease in cellular oxygen delivery and tissue oxygenation (18). Treatment of patients who had arterial hypertension and hypercholesterolemia with calcium antagonists, cholesterol decreasing agents, and low calorie diet led to normalization of the blood pressure and lipid parameters, as well as to normalization of the RBC elongation index (20).

Our data demonstrated a significant decrease in RBC deformability at the end of the 90-min shock period prior resuscitation as well as after volume resuscitation (reperfusion phase). Restoration of a normal arterial pressure did not improve RBC deformability, which remained low through the 6 h of the postresuscitation period. These RBC deformability changes were primarily observed only at low shear stresses less than 1.0 Pa. In LORCA, RBC are exposed to temperature and shear stresses similar to those observed in the circulation. The spectrum of shear stresses used in this device varies from 0.30 to 30 Pa. In vivo studies performed by different investigators have documented that shear stresses along the microcirculatory bed are close to the values mentioned above (29–31). The lowest shear stresses have been found in true capillaries (0.8–3 Pa) and venules (0.5–2 Pa). In arterioles, shear stress is significantly higher and ranges from 8 to 10 Pa to 20 to 30 Pa in shunting arterioles. Based on these data, we conclude that RBC deformability changes found at low shear stresses can have substantial impact on capillary and postcapillary blood flow.

Our results are consistent with the data of Baskurt et al. (6), who demonstrated a significant decrease in RBC EI in human and experimental sepsis only at shear stresses lower than 2 Pa (0.50 and 1.58 Pa). These authors postulate that a greater degree of RBC mechanical alteration is required to cause differences detectable at higher shear stresses. Our morphological data indirectly confirm this hypothesis because we observed a significant increase in the percentage of both reversibly and irreversibly changed RBC morphology after hemorrhagic shock. However, in absolute values, the percentage of irreversibly changed RBC did not increase dramatically and approached just 3% of the total RBC population. These results indicate that the population of RBC with the lowest levels of deformability (irreversibly damaged cells) was not large and might not result in deformability changes that can be detected at high shear stresses. On the other hand, Hardeman and Ince (32) postulated that different types of RBC deformability defects manifest themselves at different portions of the deformation curve. For example, they showed pronounced differences in the deformability of density (age) separated normal RBC and RBC with hereditary elliptocytosis only at low shear stresses, whereas in patients with Malaria tropica, significant deformability changes were found at all levels of shear stress. Patients with coronary artery disease, concomitant arterial hypertension, and diabetes also demonstrated severe RBC deformability disorders both at low and high shear stresses (33).

Taken together, our deformability and morphologic results indicate that, similar to deformability changes, RBC shape alterations can be detected at the end of shock and that they persist during 6 h of the postresuscitation period. RBC shape transformation was mainly of the reversible echinocyte type. It should be mentioned that our experimental design did not allow us to determine exactly when shock-induced shape alterations started because the first blood sample was taken for morphologic examination after 90 min of shock. However, our previous studies in a canine model of rapid hemorrhage (34) showed that significant RBC shape alterations already existed after 10 min of massive blood loss.

In our previous study (35) devoted to the influence of storage on RBC rheological properties, we showed tight correlation between the percentage of abnormally shaped RBC (reversibly and irreversibly changed) and the deformability index measured by Reid filtration technique. In the present investigation, performed by means of LORCA, this phenomenon has been confirmed in a trauma/hemorrhagic shock model. A tight relationship between RBC shape and deformability has also been confirmed by in vitro experiments (36) in which chemically induced discocyte-echinocyte and discocyte-stomatocyte RBC shape transformation resulted in the decreased RBC deformability. However, which of these alterations are primary and which are secondary remains to be determined. La Celle et al. (37), using a micropipette technique, demonstrated that in the early stages of blood storage, some normal-appearing biconcave disc-shaped RBC had decreased deformability. These results indicate that alterations of membrane deformability can be present prior to visible changes in cell shape. Thus, further work on the causal relationship between RBC shape changes and impaired deformability is required. As far as methods of RBC deformability measurements are concerned, the majority of them, including LORCA, provide the value of the mean deformability. The latter may be decreased by a slight overall deformability reduction or by the presence of a small fraction of very rigid cells. This small fraction itself can cause severe microcirculatory failure. A distinction between these two cases became possible with the recent invention of an automated rheoscope, which is able to perform RBC deformability distribution and reveal subpopulations of normal and less deformable cells (38).

Of potential clinical importance, our data showed that in the subgroup of decompensated rats, which had a 50% mortality rate in the postshock period, RBC deformability at the end of shock was significantly lower compared with the compensated animals, all of which survived the experimental period. This observation indicates an association between impaired RBC deformability and an adverse outcome. Furthermore, these results are consistent with the clinical data of Langenfeld et al. (39) obtained in patients with trauma, where the authors demonstrated that changes in deformability after trauma preceded the development of infectious complications by several days and occurred significantly earlier than the usual clinical indicators of infection, such as fever and leukocytosis. The fact that not only different species of animals (40), but different representatives of the same specie (41,42) may have different tolerance and variability in hemodynamic response to hemorrhage has been discussed in the literature. One of the explanations is a difference in sympatic nerve activity (43). Whether there exists a genetic predisposition to this phenomenon needs to be determined.

In summary, we have demonstrated that hemorrhagic shock causes RBC shape alterations and significant decreases in RBC deformability that persist in the postshock period. Because rigid RBCs with altered geometry do not traverse the capillary network well and are therefore less efficient in delivering oxygen to tissues, changes in RBC deformability may exacerbate the tissue and organ hypoxia initiated by trauma-hemorrhage.

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Keywords:

Erythrocytes; shock; hemorrhage; ektacytometry; scanning electron microscopy

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