Platelets are vital in hemostasis by forming temporary plugs and participating in the clotting reaction. Thromboembolism and bleeding are characterized by qualitative and quantitative platelet dysfunction and are important complications in asphyxiated newborns (1, 2). Disseminated intravascular coagulation is a thrombotic-hemorrhagic syndrome associated with platelet dysfunction and consumption that can occur in several clinical disorders including asphyxia, acidosis, and respiratory distress syndrome (3, 4).
Platelet function during hypoxia and reoxygenation has not yet been studied extensively. Platelets are activated during hypoxia and reoxygenation, and free radicals are associated with this. Caccese et al. have shown that platelets exposed to anoxia and reoxygenation intrinsically generate superoxide anions and hydroxyl radicals (5). These radicals can activate arachidonic acid metabolism with the production of thromboxane (Tx) A2 and subsequent activation of platelets (4, 5). Furthermore, hypoxia and reoxygenation are associated with increases in the plasma activities of matrix metalloproteinases (MMPs) 2 and 9 (6, 7), which play an important role in the regulation of platelet aggregatory function (8, 9). Platelet activation and dysfunction may be related to thromboembolic complications seen in asphyxiated newborns.
Reactive oxygen species and oxidative stress in signal transduction may be involved in certain neonatal diseases, and sick newborns are susceptible to oxidant stress when they transit from a low-oxygen to a high-oxygen environment (10). Interestingly, both clinical and animal studies reveal significantly higher oxidative stress, thus indicating more reactive oxygen species generated, in 100% oxygen resuscitated newborns compared with 21% oxygen resuscitation (11). Although there is insufficient scientific evidence to support the use of 100% over 21% oxygen during resuscitation (12), guidelines for neonatal resuscitation recommend that 100% oxygen should be used for the resuscitation of asphyxiated term newborns (13).
We recently reported the platelet aggregatory response to collagen stimulation after life-threatening hypoxia and reoxygenation with 21% or 100% oxygen in an acutely instrumented animal model of neonatal asphyxia (7). Platelets were activated with increased plasma TxB2 levels, and platelet aggregation was impaired similarly in both reoxygenated groups (7). It is uncertain if the platelet lesion is a fundamental hypoxia-reoxygenation pathology and if less than 21% oxygen concentration would be beneficial for the platelet function. Recently, hypoxic gas mixture of 17% to 19% oxygen concentration has been used as a strategy to support the cardiopulmonary hemodynamics in children with congenital heart disease (14). It is speculated that the use of hypoxic gas mixture may generate less oxygen-derived free radicals, leading to lowered oxidative stress upon reoxygenation. It is uncertain if hypoxic resuscitation will lead to less platelet activation.
The objective of this research was to study the temporal changes in platelet aggregatory function during hypoxia and reoxygenation in the newborn using a survival model of newborn piglets. We hypothesized that reoxygenation with high concentration of oxygen in hypoxic newborn piglets would activate platelets, as indicated by the plasma levels of TxB2 and MMPs, and lead to collagen-stimulated platelet aggregatory dysfunction, compared with the use of low oxygen concentrations.
MATERIALS AND METHODS
This study conforms to the standard of the Canadian Council on Animal Care. The hypoxia-reoxygenation model used has been approved by the University of Alberta Health Sciences Animal Welfare Committee.
Mixed-breed piglets 1 to 4 days of age and weighing 1.7 to 2.5 kg were used for this experiment.
Anesthesia was initially induced and maintained with inhaled halothane 2% to 5%. Piglets were endotracheally intubated and mechanically ventilated at a rate of 12 to 20 breaths per minute at pressures of 16/4 cm H2O. Via a neck incision, a 5-F silicone umbilical catheter (NeoCare, Klein-Baker Medical, San Antonio, Tex) was inserted into the left external jugular vein for the administration of medications and fluids. Another silicone umbilical catheter (5F) was inserted into the left common carotid artery for mean blood pressure monitoring and blood sampling. At the end of the surgical procedure, the neck incision was closed with sutures.
Sedation and analgesia were maintained by intravenous midazolam (0.1 to 0.2 mg/kg/h) and fentanyl (5 to 15 μg/kg/h), respectively, and muscle relaxation by pancuronium infusion (0.05 to 0.1 mg/kg/h), additional IV boluses of fentanyl (10 μg/kg), and pancuronium (0.1 mg/kg) given as needed. A dextrose-saline solution was infused at a rate of 10 mL/kg/h to maintain glucose levels and hydration. Fractional inspired oxygen concentration (FiO2), measured by an Ohmeda 5100 oxygen monitor (Madison, Wis), was maintained at 0.21 to 0.23 for oxygen saturation between 90% and 95% by a pulse oximeter (Nellcor, Hayward, Calif). Heart rate and blood pressure were continuously monitored with a Hewlett Packard 78833B monitor (Hewlett Packard Co, Palo Alto, Calif). The rectal temperature was maintained between 38.5°C and 39.5°C using a heating pad and an infrared heating lamp.
After surgery, the piglet was allowed to recover until baseline hemodynamic measurements stabilized. Stability was defined as hemodynamic measurements (heart rate and blood pressure) within 10% of the postanesthetic values. Arterial blood gases were performed regularly to ensure pH 7.35 to 7.45, PaO2 of 60 to 80 mmHg, and PaCO2 of 35 to 45 mmHg, determined by a blood gas analyzer (ABL 500, Radiometer, Copenhagen, Denmark).
Normocapnic hypoxia was initiated with the inhalation of 15% oxygen to obtain a PaO2 of 25 to 40 mmHg. After 2 h of hypoxia, the piglets were reoxygenated for 1 h with either 18%, 21%, or 100% oxygen followed by 21% oxygen for 2 h (n = 8-9 in each hypoxic-reoxygenated group). Five piglets in the control group underwent surgery and were mechanically ventilated with 21% to 23% oxygen with no hypoxia and reoxygenation. After the hypoxia and reoxygenation procedure and at the similar time of the control group, the piglets were extubated to spontaneous breathing and were allowed to recover for 4 days. No intervention was done during this period.
At the end of the study, the piglets were euthanized with an intravenous injection of pentobarbital (100 mg/kg).
Measurement of platelet aggregatory function
Based on our previous report (7), arterial blood samples (2 mL, in 3.15% sodium citrate, 9:1, v:v) were drawn for platelet aggregatory function and the determination of platelet count at baseline, after 3 h of reoxygenation, and on days 2 and 4 of recovery. Collagen-stimulated whole blood impedance aggregation meter (Chronolog Aggregometer, Chrono-log Corp, Havertown, Pa) was used to measure the platelet aggregation. The platelet aggregation was measured by the impedance between electrodes in the diluted whole blood stimulated by the agonist (collagen) at 37°C over 6 min and was expressed in ohms. Collagen concentrations of 2, 5, and 10 μg/mL (10 μL) were added to 495 μL of citrated whole blood diluted with normal saline (1:1, v:v) with maximum aggregatory response induced at 10 μg/mL based on Chronolog guidelines and our laboratory experience (0-15 μg/mL). We also determined the median effective concentration of collagen (EC50; collagen concentration required to induce 50% of maximum aggregatory response). Platelet count was measured using a hematology analyzer (MicroDiff 16, Coulter, Hialeah, Fla).
Determination of plasma Tx B2 levels, and MMP-2 and MMP-9 activities
Plasma was prepared by centrifugation at 10,000g for 15 min and stored at −80°C for subsequent biochemical analysis. Tx B2 is a stable metabolite of TxA2, which is a marker of platelet activation; and the plasma levels of this eicosanoid were assayed using a commercially available immunoassay kit (R&D Systems Inc, Minneapolis, Minn). The determination of P selectin in pigs was not attempted because of uncertain cross-reactivity between the porcine and human P selectin.
MMP-2 and MMP-9 activities in plasma were studied by gelatin zymography asdescribed previously with some modification (7). Briefly, the samples were mixed with 6× sample buffer and electrophoresed on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (0.75-mm thickness) containing gelatin (2 mg/mL) using mini-PROTEAN 3 system (Bio-Rad, Hercules, Calif). Each sample (containing 20 μg of plasma protein) was analyzed in duplicate. Human recombinant MMP-2 and MMP-9 were used as standards. After electrophoresis, the gels were washed 3 times (20 min each) in 2.5% Triton-X 100. The gels were incubated in the incubation buffer (0.05 mol/L Tris-HCl, pH 7.5, containing 0.01 mol/L CaCl2, 0.2 mol/L NaCl, and 0.05% NaN3) at 37°C for 20 h. The gels were stained with 0.05% Coomassie brilliant blue G-250 in 25% methanol and 10% acetic acid for 2 h by shaking at room temperature and then destained with 4% methanol and 8% acetic acid for 30 min. Finally, the gels were rinsed in distilled water before drying between sheets of cellophane. The zymograms were scanned using PowerLook 1000 scanner (UMAX, Dallas, Tex), and the gelatinolytic bands were analyzed using Quantity One one-dimensional analysis software (Bio-Rad). With the plasma protein concentration assessed by bicinchoninic acid assay (Sigma, St Louis, Mo) using bovine serum albumin as a standard, MMP activity was expressed in arbitrary units and compared with its respective baseline level.
Data are expressed in mean ± standard error of mean. Differences between and within groups were compared using 1-way analysis of variance (ANOVA) or ANOVA on ranks (Kruskal-Wallis) for parametric and nonparametric variables, respectively. We used the Fisher least significant difference or the Dunn method for post hoc testing if required. A statistical software (SigmaStat version 2.0, Jandel Corporation, San Rafael, Calif) was used. Significance was defined as P < 0.05.
Baseline hemodynamic and blood gas measurements are shown in Table 1. There were no significant differences in platelet aggregatory response and platelet counts at baseline between groups.
By 120 min of alveolar normocapnic hypoxia, piglets were hypotensive (mean blood pressure, 47-48 mmHg) and acidotic (mean pH, 7.19-7.24) with mean PaO2 of 30 to 35 mmHg (Table 1). The animals recovered upon resuscitation with the 1-h PaO2 of 36 ± 1, 61 ± 5, and 371 ± 12 mmHg in the 18%, 21%, and 100% reoxygenated groups, respectively. The reoxygenated groups were not different regarding the hemodynamic and blood gas measurements at 3 h of reoxygenation (data not shown).
Platelet aggregatory function and platelet counts
The control animals showed no significant change in platelet aggregatory response to 2 to 10 μg/mL collagen stimulation over 4 days of the experiment (versus baseline) (Fig. 1A).
In piglets resuscitated with 100% oxygen, collagen-induced platelet aggregation at 3 h and on day 2 of recovery was elevated when stimulated with 10 μg/mL compared with baseline (44 ± 5, 41 ± 4 vs. 26 ± 3 Ω, respectively, P < 0.05). This was shown by an upward shifting of the concentration-response curve at 3 h and day 2 without a significant difference in the EC50 (Fig. 1B, Table 2). Modest but nonsignificant increases in the platelet aggregatory response were found when stimulated with 2 or 5 μg/mL collagen at 3 h and day 2. On day 4, platelet aggregatory response normalized with no difference from baseline.
In the 18% and 21% reoxygenated groups, there were no significant changes in platelet aggregatory response during the experiment compared with baseline (Fig. 1, C and D).
Platelet counts did not change significantly during the experiment and were not different between groups (Table 2). The incidence of thrombocytopenia (platelet count < 150 × 109/L; n = 3, each in different groups) was not different between groups.
Plasma TxB2 levels
After 2 h of hypoxia, plasma levels of TxB2 were not different from baseline or between groups (Fig. 2). Three hours after resuscitation, plasma TxB2 levels increased significantly from baseline in the 100% group but not in the 18% and 21% reoxygenated groups. The increase in the 100% reoxygenated group was 3.8-fold, and this modest increase was not different between groups. The plasma TxB2 then decreased to levels not different from baselines at day 4 with no differences between groups.
Plasma MMP-2 and MMP-9 activities
Gelatinolytic activities were detected at 72 and 92 kDa and were identified as MMP-2 and MMP-9, respectively (Fig. 3). In control piglets, plasma MMP-2 activities increased (139 ± 12% of baseline) and MMP-9 activities decreased (51 ± 7% of baseline) on day 4, resulting in no significant changes in total MMP-2 and MMP-9 activity throughout the study period (119 ± 13% of baseline). After 2 h of hypoxia, plasma MMP-2 activities were lower than the baseline (79%-87% vs. 99% in controls, P < 0.05). At 3 h of reoxygenation, plasma MMP-2 activities increased to levels not different from that of controls. Piglets in the 100%, but not the 18% and 21%, reoxygenated group had significantly higher MMP-2 activities than the respective baseline activities. On day 4, the plasma activities of MMP-2 in the 21% and 100% reoxygenated groups were higher than baseline, whereas that of the 18% reoxygenated group was not different from baseline). The plasma activity of MMP-9 on day 4 was modestly lower than baseline in the 18% (P = 0.06), but not the 21% and 100%, reoxygenated group.
Annually, 3% of newborns experience asphyxia and require resuscitation, with asphyxia-related deaths varying from 0.7 to 1.2 million (15, 16). Although intraventricular hemorrhage and serious neurological morbidity have been associated with neonatal thrombocytopenia, prolonged bleeding time, and a diminished platelet aggregatory response, thromboembolic complications such as stroke and central venous thrombosis may also occur after hypoxia (17-19). Inthis study, we found increased plasma markers of platelet activation (TxB2 and MMP levels) along with a transient increase in platelet aggregatory function after hypoxia and reoxygenation with 100%, compared with 18% and 21%, oxygen. Although the clinical implication of ex vivo platelet function is uncertain, our finding is interesting because thromboembolism complications are often seen in hypoxic sick newborns.
This study is the first to examine the temporal changes in platelet aggregatory function during recovery after hypoxia in intact neonatal animals. We compared the platelet aggregatory function of hypoxic neonatal piglets resuscitated by hypoxic, normoxic, or hyperoxic gas (18%, 21%, and 100% oxygen, respectively) to assess the effect of oxidative stress on platelet activation. Although there are controversies over the use of 21% or 100% oxygen in neonatal resuscitation, it is apparent that 21% reoxygenation causes less oxidative stress than 100% reoxygenation (20). The use of hypoxic gas mixture of 17% to 19% oxygen has been studied in the resuscitation of newborn piglets (21) and recently used as a strategy to support the cardiopulmonary hemodynamics in children with congenital heart disease (14). Here we demonstrated a transient ex vivo aggregatory dysfunction of platelets in hypoxic newborn piglets resuscitated with 100% oxygen. The transient upward shifting of the collagen-aggregatory response curve may suggest an upregulation of the signaling pathway. We did not find any significant changes on the platelet activation and aggregatory function in the 21% and 18% reoxygenation groups. Studies using even lower oxygen concentration seem interesting in order to achieve significantly less oxidative stress during resuscitation, but this may not be feasible in resuscitation as evidenced by the suboptimal hemodynamic recovery in asphyxiated piglets (21).
Oxidative stress occurs when the production of free radicals exceeds the capacity of the body's antioxidative defense. These free radicals can alter lipids, proteins, and DNA (22). Hypoxia and reoxygenation have been associated with multiple organ damage including the kidneys (23), myocardium (24, 25), intestine (26), brain (27), and recently, platelet (7), which can be injured by the oxidative stress upon reperfusion (28). Vento et al. found that the newborns resuscitated with room air had lower plasma levels of oxidative stress (oxidized glutathione) at 3 and 28 days after birth than those with 100% oxygen (11, 20). It is technically challenging to study the intraplatelet oxidative stress. However, similar to our previous report (7), here we confirmed the higher level of oxidative stress-related mediator (MMPs) and the associated platelet dysfunction when 100% oxygen was used. Although the most appropriate oxygen concentration to be used during neonatal resuscitation remains controversial, some authors believe that there is sufficient data to conclude that 100% oxygen should not be given routinely anymore (29). Indeed, the neonatal resuscitation program has recently revised the guideline on oxygen concentration accordingly (13).
We speculate that the platelet dysfunction in the 100% reoxygenated group is due to the activation of platelets as a result of increased production of oxygen free radicals during reoxygenation. An extra control group subjected to 100% oxygen in the absence of a prehypoxic period may help understand if our observations are derivable from a hyperoxic-inflammatory model or a hypoxic-reoxygenation model. Oxygen free radicals can activate arachidonic acid metabolism with the production of TxA2, a strong agonist of platelet activation and a vasoconstrictor (4, 5). In this study, we found significantly elevated plasma levels of TxB2 (a marker of platelet activation) and MMP-2 (a mediator of platelet aggregatory function) in the 100% reoxygenated group. Further investigation to examine other markers of oxidative stress such as oxidized glutathione, cellular antioxidants, and peroxidation products would help establish a causal relationship between oxygen free radicals and platelet activation after hypoxia and resuscitation with high oxygen concentration.
Studies have shown that MMP-2 increases and MMP-9 decreases platelet aggregation (8, 9, 30). Although the MMPs can be activated by oxidative stress, the differential effects of MMP-2 and MMP-9 on platelet aggregation during hypoxia-reoxygenation are uncertain as observed in our study. Nonetheless, we observed an increase in the aggregatory response associated with the increased plasma TxB2 levels and MMP-2 activities in the 100% reoxygenated piglets.
Thrombocytopenia is the most common quantitative hematological abnormality leading to bleeding problems in the neonatal period (3). Compared with healthy infants, sick newborns often have reduced platelet production capacity. Perinatal asphyxia is among the common causes for early-onset thrombocytopenia (<72 h from birth). However, we did not observe any significant decrease in platelet count during the recovery period after hypoxia. Only 3 animals had thrombocytopenia. We speculate that the lack of significant decrease in platelet count may be related to the moderate degree of hypoxia as shown by the mild metabolic acidosis that resulted.
In hypoxic newborns, central lines are often used; and this may be associated with the formation of microthrombi and the subsequent development of consumptive coagulopathy because of the activation of fibrinolytic pathway and platelets with dysfunction (31). Early recognition and treatment help in reducing morbidity and mortality due to disseminated intravascular coagulopathy (30, 32). Understanding the pathogenesis should help improve patient management and reduce the occurrence of platelet dysfunction. We believe that the increased risk of thromboembolism after hypoxia is at least in part related to the hypoxia-reoxygenation process.
The association between hypoxia-reoxygenation and plateletdysfunction has not been investigated extensively. In an acutely instrumented newborn animal model of life-threatening hypoxia (2 h), we recently compared the effect of reoxygenation with 21% and 100% oxygen on the platelet aggregatory function (7). The impaired platelet aggregatory function was similar despite higher plasma levels of TxB2 and MMP-9 in the 100% reoxygenated piglets. In contrast, in this study, we have shown increased platelet aggregatory response, without altered platelet count, along with platelet activation (increased plasma TxB2 and MMP-2 levels). We believe that this difference in platelet response could be explained by the differences in the degree of hypoxia and study design. The previous experiment involved a severe life-threatening degree of hypoxia (FiO2 down to 0.10) with mean pH of 7.02 to 7.05, severe cardiogenic shock, and hypotension. However, in this survival study, piglets were moderately hypoxic, with mean pH of 7.19 to 7.24 and mild hypotension. Interestingly, Akahori et al. demonstrated the reduction in the in vitro platelet aggregation due to energy depletion at severe hypoxia (33), whereas Lehman et al. observed increased platelet aggregation with elevated markers of platelet activation in healthy volunteers exposed to mild alveolar hypoxia (34).
We should be cautious of translating the findings into clinical situations in human newborns, and this study was not powered to examine the practical consequences (clinical thromboembolism) of reoxygenation. Indeed, the platelet function might change within minutes or hours after hypoxia, resulting in thromboembolic complications after asphyxia. In our study, we measured platelet aggregatory response under the ex vivo condition; these results may not represent the actual response in vivo. In the asphyxiated human newborn, there are various factors including medications that can influence the platelets and coagulation pathway. The effect of systemic hypoxia on platelet aggregatory response in newborn piglets may depend on the degree of hypoxia induced. In our studies, we used a moderate degree of hypoxia to make survival for several days possible. The effect of severe hypoxia on platelet function will not be the same, as evidenced by the findings in our acutely instrumented piglets. Although there are reports justifying the use of plasma levels of the markers for platelet aggregation to interpret the platelet function (35), the plasma level may be derivable from other tissue elements. This is the challenge that we faced in the studies with newborn subjects because of the low sample amount resulting in very few platelets available for assay. Negative findings in this experiment, particularly in regard to the difference between groups, should be interpreted cautiously because the power of the performed tests is not optimal because of a small sample size, which was estimated to be 20 to 67 animals per group. Nonetheless, the appropriate oxygen concentration used in neonatal resuscitation requires further investigation including the multiorgan effects in surviving models.
This study provides additional evidence that caution should be taken upon resuscitation of moderately hypoxic newborns with 100% oxygen because this is associated with a higher degree of platelet activation than when a low oxygen concentration is used.
We sincerely thank Grace Chan, RN, BScN, for the assistance in the experimentation.
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