Poor perfusion and decreased oxygen transport to organs such as the intestinal tract often occur in neonates with asphyxia (1). Martin-Ancel et al. (2) evaluated a series of 72 asphyxiated term babies and found that 29% had gastrointestinal involvement. Animal studies have extensively described the redistribution of blood flow during asphyxia (3-6).
In fetal sheep with asphyxia (25 min), there was a persistent mesenteric hypoperfusion associated with increased vascular resistance upon reoxygenation (7-9). Latent gut perfusion was restored posthypoxia only when mesenteric vascular resistance decreased and not when blood pressure increased (8). However, our previous study of hypoxia (2 h) on the neonatal piglet mesenteric circulation has shown that upon reoxygenation, blood flow increased for 5 to 15 min to levels well above normoxic baseline, with a subsequent decline to baseline values with decreased vascular resistance (10).
Current treatments for postasphyxial shock include catecholamines, such as dopamine and epinephrine, which at high doses can further increase peripheral vascular resistance. Particularly, the catecholamine treatment may stress tissue oxygenation without significantly increasing mesenteric blood flow or oxygen delivery (11, 12).
Milrinone is a specific phosphodiesterase III inhibitor that increases cardiac output and also produces vasodilatation secondary to increased intracellular cyclic adenosine monophosphate concentration. The literature on the effect of milrinone in the gastrointestinal system is sparse. Studies of the isolated stomach wall of the guinea pig incubated with milrinone have shown no adverse effect on gastric ion transport (13). Further studies on adults after cardiopulmonary bypass studies confirmed that low-dose i.v. milrinone (0.25 μg/kg per min) did not negatively impact gastric pH, an indirect measure of gastric mucosa perfusion (14). Milrinone use in neonatal patients is predominantly for the improvement of a low cardiac output state (15, 16). However, there is no literature on how milrinone affects neonatal intestinal hemodynamics.
Using a swine model of neonatal hypoxia-reoxygenation (H-R), we aimed to examine the dose-dependent effects of milrinone on mesenteric hemodynamics. To investigate if the hemodynamic changes have any effect on the small intestine, we also examined the intestine regarding the effects on oxygen transport, glutathione levels, and features of reoxygenation injury. We hypothesized that milrinone would dose-dependently increase intestinal blood flow after H-R with a decrease in vascular resistance in newborn piglets.
Mixed breed piglets 1 to 3 days of age and weighing 1.5 to 2.3 kg were obtained from a local farm on the day of experimentation. The experimental protocol was approved by the University of Alberta Health Sciences Animal Welfare Committee.
The piglets were initially anesthetized with 5% halothane and maintained at 2% to 3%. Inhalational anesthesia was discontinued once mechanical ventilation via a tracheostomy was initiated. Anesthesia, analgesia, and paralysis were maintained with i.v. midazolam (0.1-0.2 mg/kg per h), fentanyl (5-15 μg/kg per h), and pancuronium (0.05-0.1 mg/kg per h), respectively, with up to two boluses of acepromazine (0.25 mg/kg). Inspired oxygen concentration was measured by an Ohmeda 5100 oxygen monitor (Ohmeda Medical, Laurel, Md) and maintained at 0.21 to 0.24 to keep oxygen saturations between 90% and 100%. Oxygen saturation was measured by continuous pulse oximetry (Nellcor, Hayward, Calif). Blood pressure and heart rate were monitored with a Hewlett Packard 78833B monitor (Hewlett Packard Co, Palo Alto, Calif). An i.v. 10% dextrose infusion at 10 mL/kg per h maintained glucose levels and hydration. An arterial line was maintained with an infusion of 0.9% isotonic sodium chloride solution at 4 mL/h. Body temperature was kept at 38.5°C to 39.5°C using an overhead warmer and heating pad.
A 5F Argyle double-lumen catheter (Sherwood Medical Co, St Louis, Mo) was inserted to the level of the right atrium via the femoral vein to deliver fluids and medications. A 5F Argyle single-lumen catheter was inserted into the right femoral artery, advanced into the infrarenal aorta, and connected to a pressure transducer to continuously measure MAP. Endotracheal intubation via a tracheostomy allowed pressure control-assisted mechanical ventilation by an infant ventilator (model IV-100, Sechrist Industries Inc, Anaheim, Calif) at a rate of 18 to 20 breaths/min and a pressure of 19/4 cm H2O.
The retroperitonium was opened through a left flank incision, and the superior mesenteric artery (SMA) was isolated with minimal dissection and encircled by a 3-mm Transonic flow probe (3SB, Transonic Systems Inc, Ithica, NY) to measure blood flow. Through a left anterior thoracotomy, the main pulmonary artery was exposed. The ductus arteriosus was ligated. A 6-mm Transonic flow probe (6SB) was placed around the base of the main pulmonary artery to monitor the blood flow as a surrogate for cardiac output. The lungs were then gently reinflated with 2 to 3 manual breaths at above pressures via the endotracheal tube. Good ventilation and perfusion were ensured.
Incisions were covered and kept moist to minimize evaporative losses. Transonic flow probes were connected to a T206 2-channel small animal flowmeter. Transonic flow probe and pressure transducer outputs were recorded and digitized by a DT2801-A analog-to-digital converter board (Data Translation, Ontario, Canada) in a Dell 425E computer equipped with custom Asyst programming software.
H-R and treatment procedure
Piglets were block randomized to 4 groups (n = 7 per group) that underwent H-R. A sham-operated group of piglets (n = 4) underwent instrumentation and stabilization without H-R or medication delivery.
After instrumentation, the animals underwent a period of stabilization for 40 min. Stability was defined as a hemodynamic measurement change ± less than 10% over 20 min, pH 7.35 to 7.45, PaO2 of 60 to 100 mmHg, and PaCO2 of 35 to 45 mmHg. Normocapnic alveolar hypoxia was initiated with the inhalation of a mixture of oxygen and nitrogen gas to obtain an inspired oxygen concentration of 0.10 to 0.15 and PaO2 of 20 to 40 mmHg for 2 h. Previous studies have determined that this degree of hypoxemia in this piglet model will produce a clinical asphyxia with severe metabolic acidosis, systemic hypotension, and a decrease in pulmonary blood flow to 40% of baseline (17, 18). Piglets were then reoxygenated with 100% oxygen for 1 h and a further 3 h of 21% oxygen. All piglets received a 10-mL/kg Ringer's lactate bolus 30 min before medication delivery. At 2 h of reoxygenation, piglets received a 2-h blinded treatment with an i.v. infusion of placebo (isotonic sodium chloride solution) or high-dose milrinone (75 μg/kg loading, 0.75 μg/kg per min infusion), mid-dose milrinone (50 μg/kg, 0.75 μg/kg per min), or low-dose milrinone (25 μg/kg, 0.25 μg/kg per min).
Medication preparation and delivery
To maintain blinding, all doses of milrinone (Milrinone Lactate; Pharmaceutical Partners of Canada Inc, Richmond Hill, Ontario, Canada) and isotonic sodium chloride solution were reconstituted in a standard volume immediately before administration. The medication was infused at 6.7 mL/kg per h for 15 min (medication loading bolus) followed by 1 mL/kg per h for the remainder of 2 h. Medication infusions were mixed by a laboratory technician uninvolved in the experiment. All medications were clear, odorless, and identified by number only.
Hemodynamic and oxygenation measurements
Systemic (heart rate, pulmonary artery blood flow, MAP) and intestinal hemodynamic parameters (SMA blood flow), blood gases, and co-oximetry were recorded at postsurgical stabilization (0 min), every 30 min during hypoxia, at 0, 10, 30, 60, and 120 min of reoxygenation, and every 30 min after medication or placebo delivery. Variables were calculated as a mean over 2 min at these specified time points.
At specified time points, simultaneous arterial and venous blood samples were taken for blood gases, hemoglobin level measurement, and co-oximetry by ABL500 (Radiometer, Copenhagen, Denmark) and OSM3 Hemoximeter (Radiometer), respectively. The mesenteric oxygen delivery (SMADO2) and estimated SMA vascular resistance index (SMAVRI) calculations were calculated using standard formulas.
Arterial blood samples (1 mL) were taken at predetermined times, centrifuged at 15,000 rotation per min (rpm) for 10 min, and the supernatant was collected and frozen at −80°C for plasma lactate determination. Less than 5% of the piglet blood volume was collected as blood work.
Small intestine tissue collection
At the end of the study, piglets were euthanized with 100 mg/kg pentobarbital i.v. Samples of distal ileum were rinsed with isotonic sodium chloride solution to remove intestinal contents and then snap frozen in liquid nitrogen and stored at −80°C, whereas another sample was fixed in 10% formalin for subsequent biochemical and histological analysis, respectively.
Lactate as a marker of anaerobic metabolism
Plasma levels of lactate were determined using a nicotinamide adenine dinucleotide (NAD) enzyme-coupled colorimetric assay. Plasma samples (15 μL) were diluted in double-distilled water (110 μL). Colorimetric microplate assay was performed with the addition of glycylglycine buffer, pH 10, NAD, double-distilled water, glutamate-pyruvate transaminase, and lactate dehydrogenase. The absorbance was read at 340 nm with a microplate spectrophotometer (Spectramax 190; Molecular Devices, Sunnyvale, Calif).
For small intestine tissue lactate determination, frozen intestine tissue (50 mg) was crushed at -80°C and then homogenized in 6% perchloric acid/0.5 mM EGTA (500 μL) on ice. After centrifuging at 11,000 rpm for 2 min at 4°C, the supernatant was weighed, and 5 M potassium carbonate was added slowly in a ratio of 1 μL:10 μL of supernatant. Precipitation on ice for 30 min was followed by centrifuging at 11,000 rpm for 2 min. The resulting supernatant was then used in substitute of plasma in the lactate assay described previously.
Intestinal tissue glutathione content
Intestinal levels of total and oxidized glutathione (GSSG) were measured using a glutathione assay kit (catalog no. 703002; Cayman Chemical, Ann Arbor, Mich). Samples of ileal tissue previously frozen at −80°C were homogenized with 1 mL/100 mg of buffer containing 0.2 M 2-N-morpholino ethanesulfonic acid, 50 mM phosphate, and 1 mM EDTA, pH 6.0. Homogenates were centrifuged at 10,000g for 15 min at 4°C, and the supernatant was collected and deproteinated with 10% metaphosphoric acid and 4 M triethanolamine, to avoid interference from sulfhydryl groups on proteins in the sample. A colorimetric microplate assay was performed by adding glutathione reductase, NADP+, and 5,5′-dithiobis-2-nitrobenzoic acid to the sample. The absorbance was measured after 25 min at 405 nm with a microplate reader (Spectra Max 190; Molecular Devices, Sunnyvale, Calif), and the total glutathione concentration was calculated from a standard curve. To measure GSSG, deproteinated samples were incubated at room temperature for 1 h with 1 M 2-vinylpyridine to completely derivatize the reduced glutathione in the sample, and the colorimetric assay was carried out as above. Glutathione redox status was obtained by calculating the ratio of GSSG and total glutathione.
Small intestine samples preserved in formalin were prepared for histological assessment using hematoxylin and eosin staining. Two independent pathologists (J.L.D. and C.G.) who were blinded to treatment group evaluated histological damage of the specimens and assigned a grade based on the Park classification of ischemic intestinal injury (19).
Plasma milrinone levels
Plasma samples previously stored at −80°C were used in a high-performance liquid chromatographic method for the determination of milrinone levels (20). Samples were mixed with internal standard and, after addition of ammonium sulfate, were extracted into ethyl acetate and back-extracted into 0.1N hydrochloric acid. Traces of ethyl acetate were removed at 45°C under nitrogen, and after pH adjustment, samples were chromatographed on a C18 column at 25°C using a mobile phase consisting of a mixture of phosphate buffer and acetonitrile. Detection of milrinone and the internal standard (0-2,000 ng/mL) was achieved by UV detection at 340 nm. Signals from the detector were collected and recorded. The limit of quantification was 5 ng/mL.
Results are expressed as mean ± SEM. Hemodynamic variables were analyzed by two-way repeated measures (RM) ANOVA followed by one-way RM ANOVA for differences within groups over time and one-way ANOVA for differences between groups at a given time point. Biochemical markers were analyzed by one-way ANOVA. If the normality test failed, ANOVA on ranks (Kruskal-Wallis) was performed. We used the Tukey or Dunn method where appropriate for pairwise comparisons in post hoc testing. Pearson moment correlation was used to determine the relationship between hemodynamic and biochemical variables. Analysis was performed using Sigma Stat (Version 2.0; Jandel Scientific, San Rafael, Calif). Significance was defined as P < 0.05.
There were no significant differences among the groups with respect to age and weight, with an average age of 2.0 ± 0.2 days and weight of 1.86 ± 0.04 kg. The mortality was 11% (4/36 piglets), secondary to surgical complications (n = 2) and severe irreversible hypoxia (n = 2).
Hypoxia and reoxygenation
At 2 h of normocapnic alveolar hypoxia, there was a metabolic acidosis with a pH 7.04 ± 0.04, PaO2 of 34 ± 1 mmHg, and arterial oxygen saturation of 34% to 40% (Table 1). The cardiac output decreased to 71 ± 4 mL/kg per min with accompanying hypotension (MAP, 28 ± 2 mmHg; Table 2). The SMA flow index (SMAFI) was reduced to 14 ± 1 mL/kg per min (41% ± 4% of the normoxic baseline; Fig. 1A). The SMADO2 decreased to 13% ± 1% of the normoxic baseline (Fig. 1B). At the end of hypoxia, the SMAVRI was not different from normoxic baseline (98% ± 8%). There were no significant differences in hemodynamic and physiological parameters during hypoxia among hypoxic groups.
Upon reoxygenation with 100% oxygen, hemodynamics immediately recovered to baseline as measured at 10 and 30 min. This recovery was not sustained, as the hemodynamic parameters gradually deteriorated over the next 90 min of reoxygenation. Arterial oxygen saturation (88%-92%), PaO2 (61 ± 1 mmHg), and pH (7.38 ± 0.01) normalized. However, MAP was decreased to 67% ± 3%; and cardiac output, to 75% ± 3% of the respective normoxic baseline (P < 0.05 vs. normoxic baseline; Table 2). After 2 h of reoxygenation, SMAFI decreased to 84% ± 5%; and SMAVRI, to 76% ± 6% of normoxic baseline (Fig. 1). No significant differences in hemodynamic parameters were found among groups before medication delivery.
After milrinone or placebo infusion
After 2 h of medication or placebo infusion, the arterial blood gas was within normal parameters (oxygen saturation, 90%-92%; PaO2, 65 ± 2 mmHg; and pH 7.40 ± 0.01). Milrinone at high dose increased and at mid and low doses maintained the cardiac output, which further deteriorated in the subsequent 2 h in hypoxic placebo controls (Table 2), illustrating a dose-related effect. There was no significant difference in heart rate and MAP among milrinone and placebo control groups after 2 h of treatment (Table 2).
Superior mesenteric circulation
After the infusion, SMAFI increased in the milrinone groups, whereas it gradually declined in the hypoxic placebo control groups (P < 0.05, high-dose milrinone group; P < 0.1, β = 0.4, mid- and low-dose milrinone groups; Fig. 1A). Superior mesenteric artery oxygen delivery was significantly increased with the high-dose milrinone infusion, with a modest effect in mid- and low-dose milrinone groups (versus hypoxic controls, P < 0.1, β = 0.5).
Although SMAFI and SMADO2 values returned to their respective normoxic baseline values after 2 h of milrinone treatment, there were significant drops in SMAFI and SMADO2 in the hypoxic placebo control group (P < 0.05 vs. normoxic baseline; Fig. 1).
In the high-dose milrinone group, there was a trend toward decreasing SMAVRI compared with the control group (P = 0.06, β = 0.6, ANOVA; P < 0.05, t test; Fig. 2A). Compared with normoxic baseline, milrinone treatment at all doses produced significantly lower SMAVRI (P < 0.05). This decrease was not found in the hypoxic control group.
Plasma milrinone levels
Plasma milrinone levels showed a significant dose-response between the groups (low, 16 ± 2 ng/mL; mid, 43 ± 4 ng/mL; high, 58 ± 5 ng/mL; P < 0.05 between the groups). Plasma milrinone levels correlated with SMAFI (r = 0.5, P < 0.05) and negatively with SMAVRI (r = −0.6, P < 0.05; Fig. 3). A negative correlation was found between the plasma milrinone levels and GSSG in the intestinal tissue (r = −0.5, P < 0.05).
Intestinal total glutathione and GSSG levels
No significant differences in total glutathione, GSSG, or GSSG/total glutathione ratio were found among treatment groups. A significant correlation was found between SMAVRI and GSSG (r = 0.6, P < 0.005) but not with the GSSG/total glutathione ratio (Fig. 2B).
Intestinal tissue lactate levels
The tissue lactate levels of the small intestine did not show a significant difference in lactate levels among groups (Fig. 4).
There were no significant differences in histology samples found between groups. Only one sample in the mid-dose milrinone group demonstrated histological changes with a small patch of denuded villi that was surrounded by normal mucosa (Fig. 5). One piglet in the low-dose milrinone group had visible intestinal pneumatosis at the time of autopsy.
During asphyxia, the neonate preserves vital organ perfusion, mainly to the heart and brain, at the expense of other organs such as the gut (1). Intestinal necrosis and injury in term neonates occur with an incidence of 0.16 to 0.71 per 1,000 live births (21). A prospective study of 72 term infants with perinatal asphyxia found that 29% of the infants had gastrointestinal symptoms consisting of any of abdominal distention, poor feeding, emesis, or mild ileus on radiographs (2).
Studies in asphyxiated newborn piglets have shown decreased blood flow and oxygen delivery, with or without an increased vascular resistance, to the gastrointestinal system during severe hypoxia (5, 6, 10, 22).
These hemodynamic changes are associated with mucosal necrosis, interstitial hemorrhage, and pneumatosis intestinalis. Improving the blood flow and reducing the vascular resistance to the gastrointestinal tract after severe hypoxia have been associated with decreased histological injury (4). In our study, we demonstrated a dose-dependent increase of SMAFI with milrinone treatment. In addition, milrinone at 0.75 μg/kg per min decreased the SMA vascular resistance compared with the gradual increase found with hypoxic placebo control treatment. This may be beneficial to the recovery of the intestine from the H-R insult.
The effects of H-R on SMAFI and SMAVRI are not clear in the literature. Videomicroscopy in acutely hypoxic piglets (30 min) has shown that decreased mesenteric flow during hypoxia coincided with a significant vasoconstrictive response, with a return to normal vessel diameter and blood flow upon reoxygenation (23). However, other hypoxic animal models demonstrate persistent intestinal hypoperfusion associated with increased vascular resistance despite reoxygenation. Restoration of gut perfusion after hypoxia occurred only when SMA vascular resistance decreased and not when blood pressure increased (7, 8).
Although high-dose milrinone infusion caused a significant increase in SMAFI over the control, our findings also support a dose-dependent response as evidenced by the modest changes at lower doses (0.25-0.50 μg/kg per min) and the positive correlation between the flow and plasma milrinone levels. The changes in mesenteric blood flow are related to the changes in regional vascular resistance, and at least in part to the concomitant changes in the cardiac output. The dose-dependent effect of milrinone on cardiac output in this study has been recently reported (18). The relationship between blood pressure, cardiac output, and intestinal perfusion during H-R is still not well defined (7, 8, 23, 24). In our study, milrinone's ability to support the intestinal perfusion coincided with an increased cardiac output without altering MAP. Thus, our study would indicate a limited role for blood pressure in restoring intestinal perfusion post-H-R.
We speculate that the small sample size of this study might have precluded us from detecting significant changes in SMAVRI (β = 0.6). High-dose milrinone infusion caused a slight decrease in SMAVRI. A significant negative correlation was observed between SMAVRI and plasma milrinone levels, supporting a dose-response effect of milrinone on the mesenteric vasculature. Milrinone's ability to inhibit phosphodiesterase and to increase cyclic adenosine monophosphate and cyclic guanosine monophosphate may augment intestinal vasodilation provided by NO during H-R.
This study demonstrated that increased SMAVRI was correlated increased GSSG. Increased GSSG may reflect the oxidative state (25) and, together with total glutathione (GSSG/total glutathione ratio), is often used as a measure of cytoplasmic redox potential and thus indicates the presence of oxygen free radicals generated by H-R in the intestine (10). Reactive oxygen species can alter the balance between NO and endothelin-1 in the intestine to favor vasoconstriction, leading to further intestinal ischemia and reperfusion injury (26). Increased milrinone levels also correlate with decreased GSSG levels. We are not certain as to the cause of this relationship between milrinone treatment and oxidative stress. Interestingly, Hayashide et al. (27) have shown that milrinone inhibited proinflammatory cytokine formation (TNF-α), thus dampening H-R injury. White et al. (28) showed a decrease in thiobarbituric acid reactive substance levels in adult patients treated with milrinone for chronic heart failure. Furthermore, the role of GSSG and oxygen free radicals such as hydrogen peroxide has been suggested in the regulation of vascular tone resulting in vasoconstriction (increased vascular resistance) in cerebral, pulmonary, and mesenteric circulations (10, 29-31).
Treatment of the clinical manifestations of shock and mesenteric hypoperfusion after asphyxia includes dopamine, dobutamine, and epinephrine. However, these catecholamines or sympathomimetics in clinically relevant doses do not increase SMAFI or decrease SMAVRI, although some may increase cardiac output with or without increased MAP (17, 32-36). In a prospective nonblinded study, 20 preterm infants with prolonged hypotension were given either 10 μg/kg per min of dopamine or dobutamine. Both blood pressure medications significantly increased the blood pressure with increased SMA blood flow velocity and decreased SMA resistive index, as measured by Doppler ultrasonography (24). This study involved preterm hypotensive infants who often have different etiologies for intestinal injury compared with asphyxiated term infants. Further studies to compare the intestinal effects of milrinone with those of currently used inotropes and pressors, such as dobutamine and epinephrine, are required.
Samples of intestine taken at the end of treatment did not show an increase in gut tissue lactate compared with hypoxic controls, indicating a lack of hypoxic stress. Furthermore, the glutathione levels were not different among the groups. Lack of intestinal damage on histology may indicate a premature analysis and should be interpreted with caution. Histopathology changes of intestine after asphyxia may take up to 24 h to detect. Intestinal necrosis and injury in term infants usually develop within days of birth, implicating perinatal events such as primary asphyxia, cardiac dysfunction, low cardiac output, and hypoxic ischemic injury as plausible pathogenic factors in these infants (21, 37-40).
Limitations of this study design include the fact that piglets, in comparison with human neonates, may metabolize milrinone differently with possible alterations in receptors and second messenger cell signaling. In this swine model of neonatal H-R, this is similar but not equivalent to neonatal asphyxia and resuscitation with 100% oxygen (41), according to the current guideline of Neonatal Resuscitation Program (42). The duration of 100% reoxygenation may be relatively long at a tertiary center but is not uncommon for resuscitations that occur in a distant region that then require medical transportation to the designated neonatal intensive care unit. Although the effect of red blood cells on the intestinal glutathione level is unknown but it is probably minimal, elegant methodological scrutiny such as the perfusion of intestinal samples to remove blood cells could have been used. Further assays of tissue oxidative markers including thiobarbituric acid reactive substances and malonyldialdehyde will be helpful. As this was an acute instrumentation study, we do not know if prolonged milrinone infusions would be detrimental or beneficial to gut perfusion or oxygen use.
In conclusion, milrinone dose-dependently increased SMA flow and oxygen delivery with significantly decreased SMAVRI at higher doses. There were no associated changes in tissue lactate, glutathione levels, or H-R histological injury in the intestine in this short-term study. Noninvasive intestinal blood flow measurement of human neonates treated with milrinone may provide future clinical data.
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