Resuscitation With 100% Oxygen Causes Intestinal Glutathione Oxidation and Reoxygenation Injury in Asphyxiated Newborn Piglets

Abstract

Objective:

To compare mesenteric blood flow, oxidative stress, and mucosal injury in piglet small intestine during hypoxemia and reoxygenation with 21%, 50%, or 100% oxygen.

Summary Background Data:

Necrotizing enterocolitis is a disease whose pathogenesis likely involves hypoxia-reoxygenation and the generation of oxygen-free radicals, which are known to cause intestinal injury. Resuscitation of asphyxiated newborns with 100% oxygen has been shown to increase oxidative stress, as measured by the glutathione redox ratio, and thus may predispose to free radical-mediated tissue injury.

Methods:

Newborn piglets subjected to severe hypoxemia for 2 hours were resuscitated with 21%, 50%, or 100% oxygen while superior mesenteric artery (SMA) flow and hemodynamic parameters were continuously measured. Small intestinal tissue samples were analyzed for histologic injury and levels of oxidized and reduced glutathione.

Results:

SMA blood flow decreased to 34% and mesenteric oxygen delivery decreased to 9% in hypoxemic piglets compared with sham-operated controls. With reoxygenation, SMA blood flow increased to 177%, 157%, and 145% of baseline values in piglets resuscitated with 21%, 50%, and 100% oxygen, respectively. Mesenteric oxygen delivery increased to more than 150% of baseline values in piglets resuscitated with 50% or 100% oxygen, and this correlated significantly with the degree of oxidative stress, as measured by the oxidized-to-reduced glutathione ratio. Two of eight piglets resuscitated with 100% oxygen developed gross and microscopic evidence of pneumatosis intestinalis and severe mucosal injury, while all other piglets were grossly normal.

Conclusions:

Resuscitation of hypoxemic newborn piglets with 100% oxygen is associated with an increase in oxygen delivery and oxidative stress, and may be associated with the development of small intestinal hypoxia-reoxygenation injury. Resuscitation of asphyxiated newborns with lower oxygen concentrations may help to decrease the risk of necrotizing enterocolitis.

In a newborn piglet model of perinatal asphyxia, our study shows that resuscitation with 100% oxygen causes increased mesenteric oxygen delivery, increased oxidative stress, and the formation of intestinal lesions similar to that seen in necrotizing enterocolitis. Resuscitation with 21% oxygen has no adverse effects in the recovery of systemic and regional perfusion and oxygenation.

Necrotizing enterocolitis (NEC) is a disease in newborns characterized by ischemic necrosis of the gastrointestinal tract.^1–3 The incidence of NEC has been increasing, and the mortality associated with it is reported from 10% to 66%.^4,5 The pathogenesis of NEC is still uncertain; however, hypoxic-ischemic injury in the perinatal period has been suggested as a likely risk factor.^2,6 Human studies have reported intestinal injury occurring in 6% to 29% of severely asphyxiated neonates,^7,8 and newborn animal models of intestinal hypoxia-reoxygenation^9–11 and ischemia-reperfusion¹² have clearly demonstrated characteristic histopathologic lesions similar to those seen in NEC.

Recently, it has been suggested that NEC may belong to the group of so-called “oxygen free radical (OFR) diseases of the newborn.”¹³ Oxygen free radicals are highly cytotoxic molecules generated during the restoration of oxygenated blood flow following ischemia or hypoxia. Perinatal asphyxia is a hypoxic-ischemic event, and with subsequent resuscitation infants are at risk for OFR-related injury to the vital organs.¹⁴ OFRs have been well established as the primary mediators of intestinal ischemia-reperfusion injury in adult animals^15–21; however, to our knowledge, there are as yet no studies examining the role of OFRs in newborn animal models of intestinal hypoxia-reoxygenation or in NEC.

Because of the risk of OFR-related injury in asphyxiated newborns during resuscitation, recent studies have investigated the effects of different oxygen concentrations used in neonatal resuscitation. Both human^22,23 and animal²⁴ studies have shown that the use of 21% oxygen is as effective or even better than 100% oxygen in the resuscitation of severely hypoxic newborns, in terms of recovery of cardiopulmonary function and organ perfusion. Furthermore, infants resuscitated with 100% oxygen had elevated blood markers of oxidative stress including an increased oxidized-to-reduced glutathione ratio,²³ which has been established as a marker for OFR-related cellular injury.^25,26

We hypothesize that OFRs generated during reoxygenation play a role in mediating intestinal injury in the newborn following asphyxia and that higher concentrations of oxygen during resuscitation may lead to an increase in OFR generation and thus predispose to increasing incidence or degree of injury. Furthermore, we hypothesize that resuscitation with lower oxygen concentrations is as effective as 100% oxygen in terms of restoration of intestinal blood flow and hemodynamic recovery.

The objectives of the present study were to compare the response of the neonatal piglet intestine to reoxygenation with 21%, 50%, or 100% oxygen following severe asphyxia. This included characterization of intestinal blood flow response to hypoxemia and graded reoxygenation, evaluation of histopathologic changes, and measurement of oxidized and reduced glutathione levels as a biochemical marker for OFR activity.

METHODS

Animals

Thirty-two newborn Yorkshire-Landrace piglets 1 to 3 days of age weighing 1.5 to 2.1 kg were obtained on the day of experimentation from a local farm. All experiments were conducted in accordance with the guidelines and approval of the Health Sciences Animal Policy and Welfare Committee of the University of Alberta.

Anesthesia

Anesthesia was induced with inhaled halothane at 5%, which was then decreased to 2% to 3%. Once mechanical ventilation was commenced, halothane was discontinued and anesthesia was maintained with intravenous fentanyl 5 to 10 μg/kg/h, midazolam 0.1 to 0.2 mg/kg/h, and pancuronium 0.05 to 0.1 mg/kg/h, with additional boluses as necessary, and acepromazine 0.25 mg/kg. Oxygen saturation was continuously monitored with a pulse oximeter (Nellcor, Hayward, CA), and heart rate and blood pressure were measured with a Hewlett Packard 78833B monitor (Hewlett Packard Co., Palo Alto, CA). Inspired oxygen concentration (F_iO₂) was measured by an Ohmeda 5100 oxygen monitor (Ohmeda Medical, Laurel, MD) and maintained at 0.21 to 0.24 for oxygen saturation between 90% and 100%. Maintenance fluids during experimentation consisted of 5% dextrose in water at 7.5 mL/kg/h and 0.9% NaCl at 2.5 mL/kg/h. Piglet temperature was maintained at 38.5°C to 39.5°C using an overhead warmer and a heating pad.

Surgical Procedure

A 5-French Argyle single-lumen arterial catheter (Sherwood Medical Co., St. Louis, MO) was inserted into the distal aorta via the right femoral artery and was attached to a pressure transducer and monitor for continuous systemic arterial blood pressure measurements. A 5-French Argyle double-lumen catheter was inserted to the level of the right atrium via the right femoral vein for the administration of medications and fluids. A tracheotomy and endotracheal intubation was performed, and pressure-controlled assisted ventilation was commenced (Sechrist infant ventilator model IV-100, Sechrist Industries Inc., Anaheim, CA) with pressures of 19/4 cm H₂O at a rate of 18 to 20 breaths/min. The retroperitoneum was opened via a left flank incision and the superior mesenteric artery (SMA) was isolated with minimal dissection and encircled with a 3-mm transit time ultrasound flow probe (3SB262, Transonic Systems Inc., Ithica, NY) attached to a Transonic T206 2-channel small animal blood flow meter for continuous measurement of blood flow. A left anterior thoracotomy in the third intercostal space was performed and a 6-mm Transonic flow probe (6SB906) was placed around the main pulmonary artery to continuously measure cardiac output. All incisions were closed or covered to minimize evaporative heat loss.

Stabilization and Monitoring

Piglets recovered from the surgical procedure until baseline hemodynamic measures were stable (change less than ±10% over 20 minutes). Preliminary arterial blood gas analysis was performed at this time and the ventilator rate was adjusted as necessary to keep the P_aCO₂ 30 to 45 mm Hg. Mean systemic arterial blood pressure (MAP), heart rate, cardiac output, SMA flow, and oxygen saturation were continuously monitored and recorded throughout the experiment. Analogue outputs of the pressure amplifiers and flow monitors were digitized by a DT 2801-A analogue to digital converter board (Data Translation, Ontario, Canada) in a Dell 425E personal computer. Software was custom written using the Asyst programming environment.

Experimental Protocol

Piglets were block randomized into four groups (n = 8 each). In 3 groups, hypoxemia was induced by ventilating the piglets with an F_iO₂ of 0.10 by increasing the concentration of inhaled nitrogen gas. The F_iO₂ was adjusted as necessary from 0.07 to 0.15 as tolerated by the piglet, to obtain a P_aO₂ of 20 to 40 mm Hg for 2 hours. This degree of hypoxemia was previously determined to consistently yield a clinically relevant degree of asphyxia, with severe hemodynamic changes in all piglets.²⁴ Following hypoxia, piglets were resuscitated with an F_iO₂ of 0.21 (21% group), 0.50 (50% group), or 1.0 (100% group) for 1 hour, followed by 0.21 for 3 hours. A control group of piglets was ventilated and oxygenated at an F_iO₂ of 0.21 for the 6 hours of study, with no period of hypoxia or reoxygenation. Hemodynamic and regional blood flow recordings were carried out at specified timepoints: baseline (0 minutes), 60, and 120 minutes of hypoxia, and 5, 10, 15, 30, 60, and 240 minutes of reoxygenation, and calculated as a mean over 2 minutes of recording. At the end of the experiment, piglets were killed with a bolus of 100 mg/kg pentobarbital.

Hemodynamic Calculations

Cardiac output and SMA blood flow were corrected for piglet weight and expressed as milliliters per minute per kilogram. SMA vascular resistance was estimated by MAP/SMA flow. Oxygen content was calculated by (1.39 × hemoglobin × O₂ saturation) + (0.03 × P_aO₂), and SMA oxygen delivery calculated by oxygen content × SMA flow.

Blood and Tissue Collection

Arterial blood samples were drawn and immediately processed for gas and acid base analysis and hemoglobin measurements (Stat Profile, Nova Biomedical, Waltham, MA) at baseline, 30 and 120 minutes of hypoxia, and 30 and 240 minutes of reoxygenation. The measured hemoglobin values did not decrease significantly during the entire experiment. Tissue was collected immediately after the piglets were killed. A sample of distal ileum, approximately 20 cm from the ileocecal valve, was collected and immediately snap-frozen in liquid nitrogen and stored at −80°C until biochemical analysis, while another sample was fixed in 10% formalin for histologic analysis.

Tissue Markers of Oxidative Stress

Glutathione

Intestinal levels of total and oxidized glutathione were measured using a glutathione assay kit (catalog no. 703002, Cayman Chemical, Ann Arbor, MI). Briefly, frozen samples of ileal tissue were homogenized with 1 mL/100 mg of buffer containing 0.2 M 2-N-morpholino ethanesulphonic acid, 50 mM phosphate, and 1 mM EDTA, pH 6.0. Homogenates were centrifuged at 10,000g for 15 minutes 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 minutes at 405 nm with a microplate reader (Spectra Max 190, Molecular Devices, Sunny Vale, CA), and the total glutathione concentration was calculated from a standard curve. To measure oxidized glutathione (GSSG), deproteinated samples were incubated at room temperature for 1 hour with 1 M 2-vinylpyridine to completely derivatize the reduced glutathione (GSH) in the sample, and the colorimetric assay was carried out as above. Reduced glutathione was calculated by subtracting GSSG from total glutathione, and glutathione redox status was obtained by calculating the ratio of GSSG:GSH.

Histologic Analysis and Grading

Intestinal specimens preserved in formalin were prepared for routine histologic evaluation using hematoxylin and eosin staining, and for scanning electron microscopy. Sections were analyzed by two independent pathologists who were not aware of the study groups, and degree of histologic damage was graded by Park's classification of ischemic intestinal injury.²⁷

Statistics

Results are expressed as mean ± standard error of the mean. The hemodynamic variables were analyzed by two-way repeated measures (RM) analysis of variance (ANOVA) followed by one-way RM ANOVA to determine differences over time compared with baseline values in each group, or one-way ANOVA to determine differences between groups at each time point (SPSS 11.0.1, SPSS Inc., Chicago, IL, and Sigma Stat 2.0, Jandel Corp., San Rafael, CA). One-way ANOVA was used to determine differences in tissue biochemical results. ANOVA was carried out as above on ranks if tests of normality or equal variances failed. Post hoc testing using the Tukey method for one-way ANOVA and RM ANOVA or Dunnett's method for ANOVA on ranks was used for pairwise comparisons. Pearson correlation analysis was used to assess the relationship between hemodynamic and biochemical variables. Histologic injury was compared using Fisher exact test. Significance was defined as P < 0.05.

RESULTS

Piglets

The median piglet age was 2 days in all experimental groups, and there were no significant differences in mean weight among the four groups. Piglets of different ages had no significant differences in baseline hemodynamic and arterial blood gas values, nor responses to hypoxia, with the exception of cardiac output, which was significantly lower at 120 minutes of hypoxia in 3-day-old piglets compared with 1- or 2-day-old piglets (P < 0.01).

Arterial Blood Gas Analysis

Severe asphyxia was achieved in our piglets after exposure to hypoxia for 2 hours, as shown in Table 1. P_aCO₂ was unchanged from baseline or control values. Upon reoxygenation, P_aO₂ was significantly higher in the 100% group compared with all other groups, and P_aO₂ in the 50% group was greater than in the 21% group or controls. At the end of the study period, there were no significant differences in pH, P_aO₂, P_aCO₂, or bicarbonate (HCO₃) among the different groups.

TABLE 1. Arterial Blood Gas and Acid-Base Status During Hypoxia and Reoxygenation

Hemodynamic Variables

Piglets subjected to hypoxia developed shock and systemic hypotension, with cardiac output and MAP both decreasing to 41% ± 2% of control values after 2 hours of hypoxemia (P < 0.001), as shown in Table 2. Heart rate did not change significantly during the study period compared with controls. Upon reoxygenation, cardiac output rapidly recovered to baseline levels in the 50% and 100% groups, and in the 21% group, cardiac output significantly increased to a maximum of 180% ± 14% of control values during the first 30 minutes of reoxygenation. MAP recovered immediately upon reoxygenation in all groups. Hemodynamic variables then remained similar to control values until the end of the study period in all experimental groups.

TABLE 2. Hemodynamic Variables During Hypoxia and Reoxygenation

Mesenteric Blood Flow

Two hours of hypoxemia caused a marked decrease in SMA flow to 34% ± 3% of control values (P < 0.001 vs. control group), as shown in Figure 1, while SMA vascular resistance remained unchanged during hypoxia (Fig. 2). Upon reoxygenation, SMA flow in the 21% group immediately increased to 177% ± 17% of the baseline value at 5 minutes of reoxygenation and remained significantly elevated at 10 and 15 minutes of reoxygenation (P < 0.05 vs. baseline), while SMA flow in the 50% and 100% groups transiently increased to 157% ± 21% and 145% ± 16% of baseline values, respectively, only at 5 minutes of reoxygenation (P < 0.05). Mesenteric vascular resistance decreased in all groups during the first 30 minutes of reoxygenation, as shown in Figure 2. There were no significant changes in SMA blood flow or vascular resistance in the control group throughout the study period.

FIGURE 1. Superior mesenteric artery (SMA) flow during hypoxia and reoxygenation with different oxygen concentrations. Control piglets (sham operated) underwent no hypoxia or reoxygenation. *P < 0.001 each hypoxic group versus control group, one-way ANOVA. ^#P < 0.05 versus baseline, one-way RM ANOVA.

FIGURE 2. Superior mesenteric artery (SMA) vascular resistance during hypoxia and reoxygenation with different oxygen concentrations. Control piglets (sham operated) underwent no hypoxia or reoxygenation. *P < 0.05 each hypoxic group versus baseline, one-way RM ANOVA. ^#P < 0.05 in the 50% and 100% groups versus baseline, one-way RM ANOVA.

Mesenteric Oxygen Delivery

Mesenteric oxygen delivery was severely impaired during hypoxemia, decreasing to 8.7% ± 2.6% of controls (P < 0.001), as shown in Figure 3. Upon reoxygenation, mesenteric oxygen delivery was significantly elevated above baseline values for the first 15 minutes of reoxygenation in the 100% group and for the first 10 minutes in the 50% group (P < 0.05). In the 21% group, oxygen delivery immediately recovered to baseline values. Mesenteric oxygen delivery in control piglets did not show any significant changes throughout the study period.

FIGURE 3. Superior mesenteric artery (SMA) oxygen delivery during hypoxia and reoxygenation with different oxygen concentrations. Control piglets (sham operated) underwent no hypoxia or reoxygenation. *P < 0.001 each hypoxic group versus control, one-way ANOVA. ^#P < 0.05 versus baseline, one-way RM ANOVA.

Small Intestinal Glutathione

Following hypoxia and reoxygenation, total glutathione levels in small intestinal tissue samples tended to decrease compared with controls, and with increasing oxygen concentrations used in resuscitation, we observed a decreasing level of small intestinal total glutathione (Fig. 4A); and reduced glutathione, (data not shown) and an increasing level of oxidized glutathione (Fig. 4B), although these differences were not statistically significant. A similar increasing trend in the glutathione redox ratio (GSSG:GSH) was observed (data not shown). Small intestinal glutathione levels in control piglets were comparable to levels previously reported in 1- and 3- day-old piglets.²⁸

FIGURE 4. Total glutathione (GSH) (A) and oxidized glutathione (GSSG) (B) in piglet small intestine after 6 hours of normoxia in sham-operated controls and after 120 minutes of hypoxia followed by 240 minutes of reoxygenation with 21%, 50%, or 100% oxygen.

Glutathione Redox Status and Mesenteric Oxygen Delivery

Mesenteric oxygen delivery during early reoxygenation correlated positively with the oxidized:reduced glutathione (GSSG:GSH) ratio (r = 0.594, 0.582, and 0.584 at 5, 10, and 15 minutes of reoxygenation, respectively, P < 0.005 for each correlation), as shown in Figure 5, and also had a significant positive correlation with the level of intestinal GSSG (P < 0.05, data not shown). Oxygen delivery during the hypoxic phase, and all other hemodynamic variables including SMA blood flow, cardiac output, or MAP had no significant correlation with glutathione levels or redox status.

FIGURE 5. Correlation between SMA oxygen delivery at 5 minutes of reoxygenation and glutathione redox status (GSSG:GSH ratio) in piglet small intestine. Regression line and correlation coefficient (r) demonstrate a significant linear relationship between intestinal oxygen delivery and GSSG:GSH ratio.

Histologic Assessment

Gross evidence of intestinal necrosis with pneumatosis intestinalis was observed in segments of the terminal ileum in 2 piglets in the 100% group, while all other specimens were grossly normal (P = 0.056, Fisher exact test, β = 0.46). Microscopically, the 2 necrotic specimens demonstrated extensive subserosal and submucosal pneumatosis, as well as areas of complete villus denudation and destruction down to the crypt level, as shown in Figure 6. All specimens were graded by Park's criteria for intestinal tissue injury, and the results are shown in Figure 7.

FIGURE 6. Histologic section (hematoxylin and eosin stain) of terminal ileum from (A) sham-operated control piglet, and piglets exposed to 2 hours of hypoxia followed by 4 hours of reoxygenation with 21% (B), 50% (C), and 100% (D) oxygen. Examples shown represent the worst histologic damage seen in each group.

FIGURE 7. Degree of intestinal tissue injury following 2 hours of hypoxia and 4 hours of reoxygenation with different oxygen concentrations. Control piglets (sham-operated) underwent no hypoxia or reoxygenation. Park's microscopic criteria for the grading of intestinal injury:²⁷ 0 = normal mucosa, 1 = subepithelial space at villus tip; 2 = extended subepithelial space; 3 = epithelial lifting along villus sides; 4 = denuded villi; 5 = loss of villus tissue; 6 = crypt layer infarction; 7 = transmucosal infarction; 8 = transmural infarction.

DISCUSSION

Our study demonstrates that severe hypoxemia in newborn piglets results in significant impairment of intestinal perfusion and oxygenation and that high oxygen concentrations used in the resuscitation of these piglets may predispose to intestinal lesions similar to those seen in NEC. Furthermore, evidence of intestinal oxidative stress as a result of high mesenteric oxygen delivery seen with higher oxygen concentrations suggest that OFRs may play a role in the pathogenesis of neonatal intestinal hypoxia-reoxygenation injury.

Necrotizing enterocolitis is the most common gastrointestinal emergency in neonates with an overall incidence of 1.1 per 1000 live births.^2,4 In 1969, Lloyd²⁹ first reported the high prevalence of asphyxia in neonates with apparently “spontaneous” gastrointestinal perforations; however, the association between perinatal asphyxia and NEC remains controversial.4,5,30–34 A recent prospective study of 72 term newborns with perinatal asphyxia found that 29% of infants had gastrointestinal manifestations, meeting the criteria of Bell et al³⁵ for suspected NEC, although none had clinical and radiologic signs of definite NEC.⁸ The pathogenesis of NEC is likely multifactorial, and intestinal hypoxia-ischemia in the neonatal period has been suggested to be an important factor.^3,6,33 A review of pathologic findings in 84 infants with NEC demonstrated ischemic necrosis in 90% of intestinal specimens from autopsy or surgery, with mucosal ulcerations, intestinal perforations, and pneumatosis intestinalis.³ Overall, the histologic damage seen in NEC is almost identical to other ischemic intestinal insults, although pneumatosis intestinalis is much more commonly seen in NEC.

While an ideal animal model of NEC remains to be established, newborn animal models of hypoxia-reoxygenation have helped to understand the pathogenesis of intestinal hypoxic-ischemic disease in the neonate. Several researchers have examined the relationship between intestinal hypoxia, blood flow, and the development of hypoxic-ischemic lesions similar to those seen in NEC.9,11,36,37 Alward et al⁹ and Karna et al¹¹ both demonstrated a significant decrease in blood flow to the gastrointestinal tract after 90 minutes of severe asphyxia in newborn piglets. Histologically, they showed evidence of patchy mucosal necrosis⁹ and findings similar to NEC including pneumatosis intestinalis in all piglets.¹¹

The results of our study are consistent with previous reports in newborn piglets,9,11,36,37 demonstrating a significant decline in SMA blood flow (34% of control values) during severe hypoxemia. We have also demonstrated that, during reoxygenation, there is a significant increase in SMA blood flow above baseline values, and furthermore that with lower oxygen concentrations used in resuscitation, the magnitude of increased SMA blood flow is greater, up to 177% of baseline blood flow with 21% resuscitation. Previous reports of intestinal blood flow during reoxygenation are contradictory, showing a hyperemic response during reoxygenation following hypoxia,²⁴ a return to prehypoxemic baseline blood flow,³⁸ or a decrease in mesenteric flow during the reoxygenation period.³⁹ While these studies^24,38,39 examined newborn piglets of similar maturity to our own, ranging from 0 to 5 days of age, the duration and severity of hypoxia varied, which may account for the different responses in SMA flow during reoxygenation. In our model, the degree and duration of hypoxia were clinically relevant, as indicated by the severity of hemodynamic changes and degree of asphyxia, which is similar to the clinical situation. More severe hypoxia of shorter duration (57 ± 6 minutes) resulting in similar clinically relevant hemodynamic and acid-base changes yielded similar changes in SMA flow upon reoxygenation, although no differences between 21% and 100% reoxygenation were reported.²⁴ This study, however, did not measure blood flow continuously and thus may have missed the differences seen between groups during early reoxygenation. Our study has further demonstrated that increased intestinal flow is a result of both decreased mesenteric vascular resistance and increased cardiac output during resuscitation. Restoration of blood flow is essential in the recovery from hypoxia-ischemia, and increased blood flow upon reoxygenation may be beneficial, as demonstrated in one previous study demonstrating that areas of the gastrointestinal tract with better perfusion following severe hypoxemia had less histologic injury.³⁶

In our study, we have demonstrated in newborn piglets subjected to hypoxia and reoxygenation the development of intestinal lesions, which are similar to those seen in NEC. Unique to our study is the demonstration that grossly necrotic lesions and pneumatosis intestinalis occur more frequently in piglets resuscitated with 100% oxygen, although this does not reach statistical significance (P = 0.056). We have shown that piglets resuscitated with 100% oxygen have a significantly higher mesenteric oxygen delivery during the first 15 minutes of reoxygenation, and interestingly that increased oxygen delivery significantly correlates with intestinal oxidative stress as measured by the glutathione redox ratio. While a definitive causal relationship has yet to be proven, we suggest that OFRs may play a role in the pathogenesis of intestinal injury in our newborn piglet model, and perhaps in NEC. It is well established that OFRs play an important role in mediating intestinal injury following ischemia-reperfusion in adult animal models,14–17,20,40–42 and it has been hypothesized that OFRs are responsible for a number of specific “oxygen radical diseases of the newborn” following perinatal asphyxia, such as chronic lung disease (bronchopulmonary dysplasia), retinopathy of prematurity, periventricular leukomalacia, patent ductus arteriosus, and NEC.^13,43 However, to our knowledge, there have been no previous studies demonstrating OFR involvement in the pathogenesis of NEC or newborn animal models of intestinal hypoxia-reoxygenation. Further studies examining the effects of antioxidant or OFR scavenger administration would help to definitively establish a causal relationship between OFRs and tissue injury following intestinal hypoxia-reoxygenation in the newborn.

In the present study, we used the intestinal glutathione redox ratio (GSSG:GSH) as a marker for OFR activity. Glutathione is one of the most important endogenous cellular antioxidants during periods of oxidative stress, in which GSH is reversibly oxidized to GSSG in the detoxification of OFRs.^25,44,45 Previous studies have established the glutathione redox status as a marker for increased oxidative stress.^23,25,26 In a prospective, randomized trial comparing the resuscitation of severely asphyxiated newborns with 100% versus 21% oxygen, Vento et al²³ demonstrated an increase in the glutathione redox ratio in blood samples at birth and 72 hours after birth in asphyxiated newborns compared with controls. Furthermore, the glutathione redox ratio was higher in infants resuscitated with 100% oxygen compared with 21% oxygen, and clinically, newborns resuscitated with 21% oxygen recovered from the asphyxial event earlier than infants resuscitated with 100% oxygen. Our present study demonstrated that intestinal GSSG:GSH ratio significantly correlated with mesenteric oxygen delivery during the first 15 minutes of reoxygenation, indicating that even a short period of exposure to high oxygen concentrations following hypoxemia can lead to increased tissue oxidative stress. The effect of this could be detrimental, as the glutathione redox status has been shown to correlate with mitochondrial DNA damage²⁶ and lipid peroxidation.⁴⁵ The absolute levels of intestinal total glutathione and GSSG, however, did not differ significantly among the experimental groups, although there was a slight trend toward an increase in GSSG and a decrease in total glutathione with increasing oxygen concentrations used in resuscitation. This may be due to the fact that intestinal tissue was collected 4 hours after reoxygenation, when oxygen delivery no longer differed between the groups and all piglets had been switched to 21% oxygen for 3 hours, thus allowing time for cellular antioxidant capacity to be replenished by the reduction of GSSG back to GSH.⁴⁴ In addition, total glutathione levels and GSSG tended to be slightly but not significantly higher in controls, which is likely the result of an overall depletion in glutathione stores during hypoxia, since glutathione synthesis is an ATP-dependent process.⁴⁴

CONCLUSION

Our study is the first to provide evidence of increased oxidative stress in the small intestine of asphyxiated newborn piglets resuscitated with 100% oxygen. Furthermore, we have shown that resuscitation with 100% oxygen may predispose to the development of small intestinal tissue injury similar to that seen in NEC, while resuscitation of severely hypoxic piglets with lower oxygen concentrations have no detrimental systemic or mesenteric hemodynamic effects. The pathogenesis of NEC is not yet fully understood, but we speculate that OFRs play a role in mediating tissue injury, similar to that seen in adult intestinal ischemic disease. Our own study supports the evidence in humans and animals that during newborn resuscitation, high oxygen concentrations should be used with caution, as potentially detrimental effects may occur in the setting of increased oxygen delivery to the vital organs.

ACKNOWLEDGMENTS

The authors thank Angela Neil for her technical assistance with the piglet experimentation, and Lynette Elder for the histologic processing of tissue samples.