The most common cause of death among preterm infants is respiratory failure, with extremely premature infants (<29 weeks estimated gestational age) at the greatest risk.1 Despite therapeutic advances such as low-pressure mechanical ventilation, high-frequency oscillatory or jet ventilation, exogenous surfactant, and extracorporeal membrane oxygenation (ECMO), morbidity and mortality remain high. Furthermore, these life-support strategies are suboptimal for extremely premature infants, as they are associated with pressure-related lung trauma and long-term morbidity.2,3 The ideal strategy for the treatment of respiratory failure in extremely premature infants would be an artificial placenta (AP), which would recreate fetal physiology by using extracorporeal life support (ECLS) for gas exchange, avoiding mechanical ventilation, and preserving fetal circulation.
In our laboratory, the AP uses venovenous (VV) ECLS with jugular drainage and umbilical vein (UV) reinfusion. We have previously demonstrated preservation of fetal circulation (patency of the ductus arteriosus), hemodynamic stability, and gas exchange for 70 hours of AP support and survival for up to 7 days.4,5 However, we had not yet described cerebral blood flow or oxygen delivery, which would be required before use of the AP in clinical practice, similar to conventional ECLS.6,7 These data can be obtained by several means. Near-infrared spectroscopy (NIRS) is an established method of measuring cerebral oxygenation in neonates on ECLS.8–10 Carotid and cerebrovascular blood flow can also be measured by ultrasonography with Doppler,11–13 and direct-contact carotid flow probes have even been used in lambs supported with veno-arterial (VA)-ECLS.14 The purpose of our study was to assess cerebral perfusion and oxygenation in this model of the AP using these technologies.
All experimental protocols were approved by our institution’s University Committee on the Use and Care of Animals. Pregnant ewes at EGA of 130–135 days (term = 145 days; n = 14) underwent laparotomy under general anesthetic. The uterus was exposed, and a hysterotomy created to expose the fetus, after which buprenorphine 0.01 mg/kg was administered subcutaneously. After local injection of 1% lidocaine, the carotid sheaths were opened by surgical cutdown. The left carotid artery was ligated distally, and a 5 Fr carotid arterial line (Arrow; Teleflex, Morrisville, NC) was placed for preductal arterial blood sampling. Heparin 100 U/kg was injected intravenously, after which the right jugular vein was cannulated with a 10–12 Fr Bio-Medicus venous cannula (Medtronic, Minneapolis, MN). One of the UVs was cannulated with a 10–12 Fr Bio-Medicus arterial cannula for reinfusion, and the remaining umbilical cord was ligated and divided. Lambs were then intubated, with the endotracheal tube filled with amniotic fluid and clamped.
The extracorporeal circuit consisted of a low resistance 0.5 m2 surface area polypropylene hollow-fiber oxygenator/heat exchanger (Capiox RX05; Terumo Cardiovascular Systems, Ann Arbor, MI), a gravity-filled peristaltic roller pump (M-pump, MC3 Inc, Ann Arbor, MI) with a flow of 50–75 ml/kg/min, and ¼-inch Tygon tubing (Saint-Gobain North America, Malverne, PA). The sweep gas was comprised of a mixture of 70% O2 and 3% CO2 with balance nitrogen, and sweep flow was maintained at 1–3 L/min.
The lambs were incubated in a dry heated waterbed to help maintain core fetal temperature of 39°C (normal lamb temperature is 38–40°C) and heated via the heat exchanger in the AP circuit. Broad-spectrum antibiotics were given. Fetal volume status was maintained with a 10% dextrose/0.225% NaCl solution at 4 ml/kg/hr. Additional boluses of colloids and crystalloids were given as needed for hypotension or low circuit flow. Lambs were transfused for hemoglobin levels <10 g/dL with blood procured from the native placenta at the time of delivery. Heparin was infused and titrated to a goal activate clotting time (ACT) of 180–200 s. Support was continued for up to 4 days, after which time the lamb was sacrificed. After sacrificed, brains were procured and examined for gross pathology. Brains were then sectioned for histopathologic analysis in the case of gross pathology.
Postoperative Data Collection
Fetal blood gases were collected hourly from the carotid artery and analyzed (ABL 505; Radiometer America, Cincinnati, OH). In addition, hemodynamics, blood glucose, temperature, and ACTs were recorded at regular intervals during AP support.
Cerebral Blood Flow and Oxygenation
During the operative instrumentation, the right carotid artery was isolated, and a 2.5 mm ultrasonic flow probe (Transonic Systems Inc, Ithaca, NY) was placed around it for continuous carotid arterial flow (CAF) monitoring. Before cannulation or ligation of the umbilical cord, baseline CAF values were collected from experimental animals while still on native placental support. These values were also recorded every 30 minutes during artificial placental support and adjusted to body weight. The ratio of CAF to mean arterial pressure (MAP) was also calculated.
A transcranial Somanetics INVOSTM Cerebral Oximeter pediatric sensor (Somanetics Corporation, Troy, MI) was placed on the center of each lamb’s head to noninvasively monitor cerebral oxygenation—these probes remained in place for the entirety of the experiment without repositioning. NIRS measures reflect venous and arterial oxygenation in a ratio of approximately 84:16.15 The regional oxygen saturation (rSO2) values were used in conjunction with the oxygen saturation (SaO2) of blood drawn from the carotid artery to calculate the cerebral fractional tissue oxygen extraction (FTOE). FTOE calculated as the ratio of brain oxygen consumption served as a relative measure of the balance between brain oxygen delivery and consumption (FTOE = [SaO2 − rSO2]/SaO2).16 Because rSO2 predominantly reflects venous SaO2, an increase in FTOE reflects higher oxygen extraction or increased oxygen consumption compared with oxygen delivery. Conversely, decreased FTOE implies decreased oxygen consumption relative to oxygen delivery.17,18 As with baseline CAF, baseline NIRS values were collected from experimental animals before cannulation or umbilical cord ligation while still on native placental support. NIRS values were subsequently recorded every 30 minutes after initiation of AP support.
Data were analyzed in each sample collection time as a separate data point. Values are expressed as mean ± standard deviation (SD). Statistical analysis was performed using SPSS (IBM, Armonk, NY), including paired t-tests and Pearson correlation coefficients. A p value of <0.05 was considered statistically significant.
Lambs were successfully supported for 55 ± 27 hours (range 10–92 hours) of support. Death was caused by cardiovascular collapse, sepsis, or elective sacrifice. Mean body weight at the conclusion of all experiments was 4.3 ± 0.6 kg (range 3.5–5.1 kg). Mean hemodynamics, carotid arterial blood gas values, and acid–base status were within the normal fetal range and stable throughout AP support (Table 1).
Baseline CAF as measured by Doppler before cannulation on native placental support was 25.1 ± 4.5 ml/kg/min, compared with 23.7 ± 7.7 ml/kg/min during AP support (p = 0.47). Cerebral oxygenation values at baseline on native placenta support were 40% ± 3%, compared with mean rSO2 during AP support of 50% ± 11% (p = 0.027). FTOE during AP support was 37% ± 13% compared to 35% ± 13% extraction with native placental support (p = 0.69). Cerebral FTOE correlated negatively with CAF (r = −0.382; p < 0.001) (Figure 1) and MAP (r = −0.425; p < 0.001) (Figure 2). Conversely, the CAF/MAP ratio correlated positively with arterial partial pressure of carbon dioxide (pCO2) (r = 0.252; p < 0.001) (Figure 3).
All lambs displayed spontaneous movement during AP support and retained baseline reflexes (corneal, suckling, and anal wink). On necropsy, there was no gross or microscopic evidence of intracranial hemorrhage in any animals. However, one lamb did have a single 5 mm area of grossly soft parenchyma in the left frontal cortex and signs of neuronal death in this region on histopathology.
We sought to establish the adequacy of cerebral blood flow and oxygenation during support with an AP in a premature lamb model. The means of assessment—NIRS as a noninvasive continuous measurement of cerebral oxygenation and carotid arterial blood flow by ultrasonography—are well established.11–13,15,19,20 During artificial placental support of 14 lambs for 10–92 hours, NIRS and CAF suggest that cerebral oxygen delivery is preserved.
Since the early days of ECLS, cerebral ischemia has been a feared complication.12,21,22 Cerebral perfusion depends on the mode of support; VA-ECLS entails ligation of the right carotid artery and jugular vein and is directly associated with temporary reduction in flow to the right middle cerebral artery (RMCA) distribution right after initiation of ECLS, but this generally returns to normal.11–13,23 VV-ECLS spares the carotid artery but requires ligation of the right jugular vein; cerebrovascular congestion from this ligation is associated with abnormalities of blood flow including decreased RMCA flow.12,24,25 Studies have not consistently shown that right-sided hypoperfusion occurs long-term nor that it is associated with specific deficits.
Lambs supported by the AP are susceptible to similar alterations in cerebral perfusion because they undergo ligation of the right jugular vein. In this particular model, it also involves ligation of the left carotid artery to allow for blood gas sampling. Cerebral blood flow in this model is therefore dependent on adequate cardiac function (as opposed to VA-ECLS, which provides an element of cardiac support in addition to gas exchange) and an intact circle of Willis. Conceptually, this places our model at the highest risk for cerebral hypoperfusion because it incorporates the drawbacks of both VA- and VV-ECLS (Figure 4). Clinical application of the AP in human infants would preserve bilateral carotid circulation because the umbilical artery would be used for arterial access. Therefore, our observation that cerebral oxygenation and CAF are preserved in this high-risk model makes the most conservative case for preservation of cerebral oxygen delivery with the AP. Whether cerebral hypertension became a clinical problem, a cephalad drainage catheter could be inserted to allow cerebral venous drainage.26
Our results indicate that CAF was near baseline values during AP support despite the ligation of the contralateral carotid artery for a monitoring line. Additionally, cerebral rSO2 values were not significantly different from baseline levels that were collected from similarly aged fetal lambs on native placental support and approximate values that have been noted in premature sheep supported by mechanical ventilation.27 In normal human neonates, rSO2 rises during the first few minutes of life before reaching stable levels.28,29 This is consistent with our observation that rSO2 was lower during native placental support, because more highly oxygenated blood is being delivered via the UVs under artificial placental support.
This observation highlights the novelty of our experimental model and findings. The effects of ECLS on cerebral blood flow and oxygen delivery in newborn lambs have been previously studied by Hunter et al.30 They reported that VV-ECLS had no impact on either variable. Similarly, carotid ligation only caused a brief decrease in right cerebral blood flow (CBF) that resolved in under 1 minute. It may not be surprising, then, that lambs in our study maintained cerebral oxygenation while undergoing VV-ECLS and contralateral carotid ligation. However, it is important to note the difference in our animal model and the ECLS configuration used by the AP. Preterm lambs maintain fetal circulation immediately after delivery, with shunts including the foramen ovale and ductus arteriosus. Although the AP does use VV-ECLS, the UV reinfusion is paramount to its design, as it preferentially shunts oxygenated blood through the foramen ovale into the left heart, which from there supplies the premature brain. Whether we were to use conventional double lumen VV-ECLS as has previously been described, oxygenated blood would preferentially cross the tricuspid valve to be ejected into the pulmonary artery, through the ductus arteriosus, and to the descending aorta, thereby bypassing the cerebral circulation.
Making use of carotid arterial O2 saturation and rSO2, we calculated FTOE levels for a better understanding of rSO2 during AP support. “Luxury perfusion” (increase in rSO2 with decrease in FTOE) has been reported in sheep models of hypoxic-ischemic cerebral injury.31 FTOE values during AP support were very close to FTOE levels on native placental support, suggesting that the study animals did not experience luxury brain perfusion. Also, we found that FTOE decreased as carotid flow increased and was inversely correlated with MAP. These findings support the theory that our AP model allows the animal to maintain appropriate cerebrovascular autoregulation.
We also found that CAF/MAP correlated positively with pCO2. This observation aligns with previous studies in normal human physiology that demonstrate a positive correlation between carotid arterial blood flow and pCO2.32,33 This finding also suggests that the CO2-based cerebrovascular autoregulation mechanism remains intact during AP support.
Our study has several important limitations. Because of the complexity of animal care and our laboratory capabilities at the time, we were limited to collecting data every 30 minutes instead of continuously. This could have caused some variation because of artifact. Also, skull thickness in premature sheep mandated placement of the NIRS probe in midline skull, likely over the sagittal sinus. This would suggest that saturations may have reflected venous blood even more than that published 84%. Because we used late-term lambs to establish our VV-ECLS AP model, our findings may not translate directly to extremely premature human infants. Furthermore, clinical application of the AP may require several weeks of extracorporeal support, and further evaluation will need to be performed in significantly premature sheep long term. We have established that a premature lamb at 118 days gestation correlates to a 24 week EGA human infant in terms of lung development.5 We therefore will need to repeat these studies in this more premature model to confirm similar findings at this earlier gestational age.
Although we demonstrated adequate cerebral perfusion with the AP, further studies will need to address cerebral function, evaluate white matter injury, and address the issue of intraventricular hemorrhage (IVH). Preliminary work in our laboratory demonstrate the feasibility of using amplitude-integrated electroencephalography to assess function and ability of postmortem magnetic resonance imaging (MRI) to evaluate brain structure, myelination, and injury.34 Although IVH has rarely been observed in our heparinized AP experiments, the sheep is not a good model for IVH because the germinal matrix is present at approximately 70 days gestation.35 Clinical translation to premature infants already at risk for IVH will require elimination of heparin and development of nonthrombogenic surfaces.36,37
Cerebral oxygenation and CAF are maintained throughout AP support with VV-ECLS. This suggests that cerebral perfusion and oxygenation are maintained and adequate during AP support.
The authors thank the Somanetics Corporation for their donation of equipment for this experiment.
1. Greenough A, Murthy VRespiratory problems in the premature newborn. Ped Health 2009.3: 241–249,
2. Sweet DG, Carnielli V, Greisen G, et alEuropean Association of Perinatal Medicine: European consensus guidelines on the management of neonatal respiratory distress syndrome in preterm infants - 2010 update. Neonatology 2010.97: 402–417,
3. Bahrami KR, Van Meurs KPECMO for neonatal respiratory failure. Semin Perinatol 2005.29: 15–23,
4. Gray BW, El-Sabbagh A, Zakem SJ, et alDevelopment of an artificial placenta V: 70 h veno-venous extracorporeal life support
after ventilatory failure in premature lambs. J Pediatr Surg 2013.48: 145–153,
5. Bryner B, Gray B, Perkins E, et alAn extracorporeal artificial placenta supports extremely premature lambs for 1 week. J Pediatr Surg 2015.50: 44–49,
6. Campbell LR, Bunyapen C, Holmes GL, Howell CG Jr, Kanto WP JrRight common carotid artery ligation in extracorporeal membrane oxygenation
. J Pediatr 1988.113 (1 pt 1): 110–113,
7. Bartlett RH, Gazzaniga AB, Toomasian J, et alExtracorporeal membrane oxygenation (ECMO) in neonatal respiratory failure. 100 cases. Ann Surg 1986.204: 236–245,
8. Liem KD, Hopman JC, Oeseburg B, de Haan AF, Festen C, Kollée LACerebral oxygenation and hemodynamics during induction of extracorporeal membrane oxygenation
as investigated by near infrared spectrophotometry. Pediatrics 1995.95: 555–561,
9. Papademetriou MD, Tachtsidis I, Elliot MJ, Hoskote A, Elwell CEMultichannel near infrared spectroscopy indicates regional variations in cerebral autoregulation in infants supported on extracorporeal membrane oxygenation
. J Biomed Opt 2012.17: 067008,
10. Tyree K, Tyree M, DiGeronimo RCorrelation of brain tissue oxygen tension with cerebral near-infrared spectroscopy and mixed venous oxygen saturation during extracorporeal membrane oxygenation
. Perfusion 2009.24: 325–331,
11. Matsumoto JS, Babcock DS, Brody AS, Weiss RG, Ryckman FG, Hiyama DRight common carotid artery ligation for extracorporeal membrane oxygenation
: cerebral blood flow velocity measurement with Doppler duplex US. Radiology 1990.175: 757–760,
12. Fukuda S, Aoyama M, Yamada Y, et alComparison of venoarterial versus venovenous access in the cerebral circulation of newborns undergoing extracorporeal membrane oxygenation
. Pediatr Surg Int 1999.15: 78–84,
13. Weber TR, Kountzman BThe effects of venous occlusion on cerebral blood flow characteristics during ECMO. J Pediatr Surg 1996.31: 1124–1127,
14. Stolar CJ, Reyes CExtracorporeal membrane oxygenation causes significant changes in intracranial pressure and carotid artery blood flow in newborn lambs. J Pediatr Surg 1988.23: 1163–1168,
15. Watzman HM, Kurth CD, Montenegro LM, Rome J, Steven JM, Nicolson SCArterial and venous contributions to near-infrared cerebral oximetry. Anesthesiology 2000.93: 947–953,
16. Toet MC, Lemmers PM, van Schelven LJ, van Bel FCerebral oxygenation and electrical activity after birth asphyxia: Their relation to outcome. Pediatrics 2006.117: 333–339,
17. Naulaers G, Morren G, Huffel SV, et alCerebral tissue oxygenation index in very premature infants. Arch Dis Child Fetal Neonatal Ed 2002.87: F189–F192,
18. Lemmers PM, Toet M, van Schelven LJ, van Bel FCerebral oxygenation and cerebral oxygen extraction in the preterm infant: The impact of respiratory distress syndrome. Exp Brain Res 2006.173: 458–467,
19. Weiss M, Dullenkopf A, Kolarova A, Schulz G, Frey B, Baenziger ONear-infrared spectroscopic cerebral oxygenation reading in neonates and infants is associated with central venous oxygen saturation. Paediatr Anaesth 2005.15: 102–109,
20. Franceschini MA, Boas DA, Zourabian A, et alNear-infrared spiroximetry: Noninvasive measurements of venous saturation in piglets and human subjects. J Appl Physiol (1985) 2002.92: 372–384,
21. Short BLThe effect of extracorporeal life support
on the brain: A focus on ECMO. Semin Perinatol 2005.29: 45–50,
22. Hofkosh D, Thompson AE, Nozza RJ, Kemp SS, Bowen A, Feldman HMTen years of extracorporeal membrane oxygenation
: Neurodevelopmental outcome. Pediatrics 1991.87: 549–555,
23. O’Brien NF, Hall MWExtracorporeal membrane oxygenation and cerebral blood flow velocity in children. Pediatr Crit Care Med 2013.14: e126–e134,
24. Taylor GA, Walker LKIntracranial venous system in newborns treated with extracorporeal membrane oxygenation
: Doppler US evaluation after ligation of the right jugular vein. Radiology 1992.183: 453–456,
25. O’Connor TA, Haney BM, Grist GE, Egelhoff JC, Snyder CL, Ashcraft KWDecreased incidence of intracranial hemorrhage using cephalic jugular venous drainage during neonatal extracorporeal membrane oxygenation
. J Pediatr Surg 1993.28: 1332–1335,
26. Skarsgard ED, Salt DR, Lee SKExtracorporeal Life Support Organization Registry: Venovenous extracorporeal membrane oxygenation
in neonatal respiratory failure: Does routine, cephalad jugular drainage improve outcome? J Pediatr Surg 2004.39:672–676,
27. Barton SK, Moss TJ, Hooper SB, et alProtective ventilation of preterm lambs exposed to acute chorioamnionitis does not reduce ventilation-induced lung or brain injury. PLoS One 2014.9: e112402,
28. Pichler G, Avian A, Binder C, et alaEEG and NIRS during transition and resuscitation after birth: Promising additional tools; an observational study. Resuscitation 2013.84: 974–978,
29. Pichler G, Binder C, Avian A, Beckenbach E, Schmölzer GM, Urlesberger BReference ranges for regional cerebral tissue oxygen saturation and fractional oxygen extraction in neonates during immediate transition after birth. J Pediatr 2013.163: 1558–1563,
30. Hunter CJ, Blood AB, Bishai JM, et alCerebral blood flow and oxygenation during venoarterial and venovenous extracorporeal membrane oxygenation
in the newborn lamb. Pediatr Crit Care Med 2004.5: 475–481,
31. Bennet L, Roelfsema V, Pathipati P, Quaedackers JS, Gunn AJRelationship between evolving epileptiform activity and delayed loss of mitochondrial activity after asphyxia measured by near-infrared spectroscopy in preterm fetal sheep. J Physiol 2006.572 (pt 1): 141–154,
32. Reivich MArterial PCO2
and cerebral hemodynamics. Am J Physiol 1964.206: 25–35,
33. Kety SS, Schmidt CFThe effects of altered arterial tensions of carbon dioxide and oxygen on cerebral blood flow and cerebral oxygen consumption of normal young men. J Clin Invest 1948.27: 484–492,
34. Griffith JL, Shimony JS, Cousins SA, et alMR imaging correlates of white-matter pathology in a preterm baboon model. Pediatr Res 2012.71: 185–191,
35. Balasubramaniam J, Del Bigio MRAnimal models of germinal matrix hemorrhage. J Child Neurol 2006.21: 365–371,
36. Major TC, Brant DO, Reynolds MM, et alThe attenuation of platelet and monocyte activation in a rabbit model of extracorporeal circulation by a nitric oxide releasing polymer. Biomaterials 2010.31: 2736–2745,
37. Handa H, Brisbois EJ, Major TC, et alIn vitro
and in vivo
study of sustained nitric oxide release coating using diazeniumdiolate-oped poly(vinyl chloride) matrix with poly(lactide-co-glycolide) additive. J Mater Chem B Mater Biol Med 2013.1: 3578–3587,