Hypothermia therapy for neonatal encephalopathy
|Hypothermia therapy for neonatal encephalopathy|
Brain hypothermia, induced by cooling a baby to around 33 °C for three days after birth, is a treatment for hypoxic ischemic encephalopathy. It has recently been proven to be the only medical intervention which reduces brain damage, and improves an infant's chance of survival and reduced disability. Hypoxic ischemic encephalopathy has many causes and is essentially the reduction in the supply of blood or oxygen to a baby's brain before, during, or even after birth. It is a major cause of death and disability, occurring in approximately 2–3 per 1000 births and causing around 20% of all cases of cerebral palsy.
A 2013 Cochrane review found that therapeutic hypothermia is useful in full term babies with encephalopathy.
Extended follow-up of trial participants
Studies have been undertaken to determine the effects of hypothermia beyond early childhood. Participants in the CoolCap, NICHD and TOBY trials were entered into extended follow-up programmes. None of these programmes have sufficient power to make confident assessments of the long-term effect of hypothermia, however even these underpowered studies give important information on whether the therapeutic effects of cooling are sustained beyond the first two years after birth.
The most significant follow-up study published so far is the assessment of the NICHD trial participants at 6–7 years. Of the 208 trial participants, primary outcome data were available for 190. Of the 97 children in the hypothermia group and the 93 children in the control group, death or an IQ score below 70 occurred in 46 (47%) and 58 (62%), respectively (P=0.06); death occurred in 27 (28%) and 41 (44%) (P=0.04); and death or severe disability occurred in 38 (41%) and 53 (60%) (P=0.03). The CoolCap study gathered data using the WeeFim questionnaire at 7–8 years of age, but only collected information on 62 (32 cooled; 30 standard care) of 135 surviving children who had had neurodevelopmental assessment at 18 months. Disability status at 18 months was strongly associated with WeeFIM ratings (P < 0.001) suggesting that the therapeutic effect persisted, but there was no significant effect of treatment (P = 0.83).
These results were not quite conclusive, as the effect in the NICHD trial appears to be on mortality rather than neurological function, but they gave considerable confidence that the therapeutic effects of hypothermia following birth asphyxia are sustained into later childhood, and the preliminary childhood outcomes of the Toby trial presented at the Hot Topics in Neonatology meeting in Washington DC in December 2013 appear to confirm persisting benefit of treatment on neurological function; these data await peer review and formal publication and if confirmed would provide the definitive data needed for evidence-based health policy decisions.
Current state of the evidence
Hypothermic neural rescue therapy is an evidence-based clinical treatment which increases a severely injured full term infant's chance of surviving without brain damage detectable at 18 months by about 50%, an effect which seems to be sustained into later childhood.
At present data relate only to full term infants, and all human studies of hypothermia treatment have so far been restricted to infants >36 weeks out of an expected 40 weeks gestation. There are both more potential side effects on the developing premature with lung disease, and there is more evident protection by hypothermia when a greater volume of complex brain is actively developing. During mid gestation to late term the fetal brain is undergoing increasingly complex progressive growth of first the mid-brain and then development of the cortex and "higher" centers. The effects of fetal asphyxia on the developing brain in sheep are dependent on gestational age with near term fetuses showing both less tolerance of asphyxia and maximal damage in the rapidly expanding cortex; while fetuses prior to the last third of development experience more extended tolerance of asphyxia with maximal effects on the growing mid-brain. The fetal sheep asphyxia model also suggests a six-hour window post asphyxia in which hypothermia will have greatest benefit.
There remains much that is unknown. Recognition of infants with marginal external signs of asphyctic damage at birth, who still develop moderate hypoxic ischemic encephalopathy would be enhanced by finding more reliable bio-markers or physiologic tests accurately predicting the risk for progressive damage. These tests could also prevent unwarranted, expensive treatment of many infants. Long-term follow-up has yet to demonstrate show persisting benefit, but available data together with an imaging study nested in TOBY also found reduced brain tissue damage in cooled infants are encouraging.
The simplicity that attracted empiricists to cooling centuries ago now makes hypothermic neural rescue with accurate patient selection a potentially transforming therapy for low-resource environments where birth asphyxia remains a major cause of death and disability. Ironically this brings back the problem of cooling infants in an environment where modern resuscitation and intensive care are not available.
Mechanisms of action
Much of what is known about the mechanisms of hypothermic neuroprotection is gathered from studies in mature and adult models. What follows uses some of these data while trying to focus on the immature brain.
Cerebral hypoxia-ischaemia results in reduced cerebral oxidative metabolism, cerebral lactic acidosis and cell membrane ionic transport failure; if prolonged there is necrotic cell death. Although rapid recovery of cerebral energy metabolism occurs following successful resuscitation this is followed some hours later by a secondary fall in cerebral high energy phosphates accompanied by a rise in intracellular pH, and the characteristic cerebral biochemical disturbance at this stage is a lactic alkalosis. In neonates, the severity of this secondary impairment in cerebral metabolism are associated with abnormal subsequent neurodevelopmental outcome and reduced head growth.
Several adverse biological events contribute to this secondary deterioration, including: release of excitatory amino acids which activate N-methyl-D-aspartate (NMDA) and amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors on neurons (30,37) and oligodendroglial precursors, accumulation of excitatory neurotransmitters, generation of reactive oxygen radicals, intracellular calcium accumulation and mitochondrial dysfunction. Whilst necrotic cell death is prominent in the immediate and acute phases of severe cerebral insults, the predominant mode of death during the delayed phase of injury appears to be apoptosis. Neuroprotective mechanisms need to interact with these mechanisms to have beneficial effect.
Newborn hypoxic-ischaemic brain injury differs from injury in the adult brain in several ways: NMDA receptor toxicity is much higher in the immature brain. Apoptotic mechanisms including activation of caspases, translocation of apoptosis-inducing factor and cytochrome-c release are much greater in the immature than the adult. The inflammatory activation is different with less contribution from polymorphonuclear cells and a more prominent role of IL-18 whereas IL-1, which is critical in the adult brain, is less important. The anti-oxidant system is underdeveloped with reduced capacity to inactivate hydrogen peroxide.
Actions of hypothermia
Mild hypothermia helps prevent disruptions to cerebral metabolism both during and following cerebral insults. Hypothermia decreases the cerebral metabolic rate for glucose and oxygen and reduces the loss of high energy phosphates during hypoxia-ischaemia and during secondary cerebral energy failure, and reduces delayed cerebral lactic alkalosis. The simultaneous increase in cytotoxic oedema and loss of cerebral cortical activity that accompanies secondary energy failure is also prevented.
Hypothermia appears to have multiple effects at a cellular level following cerebral injury. Hypothermia reduces vasogenic oedema, haemorrhage and neutrophil infiltration after trauma. The release of excitatory neurotransmitters is reduced, limiting intracellular calcium accumulation. Free radical production is lessened, which protects cells and cellular organelles from oxidative damage during reperfusion. In addition mild hypothermia may reduce the activation of the cytokine and coagulation cascades through increased activation of suppressor signalling pathways, and by inhibiting release of platelet activating factor.
Many of the effects induced by mild hypothermia may help to reduce the number of cells undergoing apoptosis. Experimental and clinical studies indicate that the number of apoptotic neurons is reduced caspase activity is lessened and cytochrome c translocation is diminished by mild hypothermia, and there may be an increase in expression of the anti-apoptotic protein BCl-2.
Many physicians over the centuries have tried to resuscitate babies after birth by altering their body temperatures, essentially aiming to animate the infant by inducing the onset of breathing. Little thought was given to brain protection, because cerebral hypoxia during birth was not linked with later neurological problems until William John Little in 1861, and even then this was controversial; Sigmund Freud, for example, famously disagreed, and when scientific studies of neonatal therapeutic hypothermia were begun in the 1950s researchers like Bjorn Westin still reported their work in terms of re-animation rather than neuroprotection. Investigators such as James Miller and Clement Smith carried out clinical observations and careful physiological experiments, but although some babies were conscientiously followed up, they were not mainly concerned with long term neurological outcome.
However, by the 1960s physicians saw hypothermia after delivery was something to be avoided. The problem of infants who failed to breathe at birth had been solved by the invention of mechanical ventilation, so any benefit cooling might have for re-animation was no longer needed, and an influential trial showed that keeping small and preterm infants warm increased survival. These results, together with observational and experimental data made it an article of medical faith for decades that babies should not be allowed to get cold.
Consequently, during the next two decades studies of neonatal hypothermia in Europe and the USA were sporadic and often unsuccessful. An interest in cooling for brain protection was beginning to emerge, but contemporary neuroscience provided few useful concepts to guide this research and little progress was made. Although across the Iron Curtain in the Soviet Union cooling was being applied empirically following birth asphyxia, the language barrier, cold war politics and the Russians' failure to carry out randomised controlled trials contributed to an almost total ignorance of this work in the West. Indeed, a group of Russian neonatologists who described hypothermic neural rescue during a visit to the Neonatal Unit in Bristol, UK, met with little interest.
In the late 1980s the development of a new set of concepts and problems led to a re-examination. A new generation of neonatal researchers were influenced by the growing evidence that protecting the brain against the effects of oxygen deprivation during labour might be possible. These researchers were aware that cooling produced powerful intra-ischaemic neuroprotection during cardiac surgery but a new concept of hypothermic post-insult neural rescue developed. This shift in thinking was possible because of at least three major new ideas that were developing at the same time: delayed post-ischaemic cell death; excitotoxicity; and apoptosis.
Delayed cell death
The first paradigm shift that affected neonatal researchers in particular was the idea that if a baby was resuscitated after cerebral hypoxia-ischaemia there was a period of time before brain cells started to die. Osmund Reynolds at University College London used the newly developed technique of Magnetic Resonance Spectroscopy (MRS) to show that the infant brain metabolism is normal in the hours after birth asphyxia and deteriorated only after a distinct delay. Robert Vannucci confirmed the effect with painstaking biochemistry, and delayed injury was also reported in neuropathological studies.
Delayed brain injury (called ‘secondary energy failure’ by Reynolds) was a critical new idea. If brain cells remained normal for a time and the mechanism of the delayed death could be unravelled, it opened the possibility of therapeutic intervention in what had previously seemed an impossible situation.
The new and transforming concept of excitotoxicity developed from the seminal experiments of John Olney and Brian Meldrum. They showed that at least some of the neural cell death caused by hypoxia-ischaemia is mediated by excess production of the excitatory neurotransmitter glutamate, and that pharmacological blockade of the N-methyl-D-aspartate receptor could provide good protection against hypoxic damage. Olney and Meldrum had shifted the paradigm, allowing researchers to think of hypoxic-ischaemic damage as a treatable disease.
However, it was still a mystery how and why cells triggered by hypoxia-ischaemia should die hours or days later, particularly when it became clear that glutamate levels were not particularly high during secondary energy failure. The next critical idea came with the discovery of programmed cell death, a novel form of cell suicide. Originally observed as a pathological appearance and named apoptosis ("falling off", as of leaves) in the 1970s, Horvitz, Raff and Evan provided a molecular understanding and showed that apoptosis could be triggered by cellular insults. The radical idea that hypoxia-ischaemia triggered a cell suicide programme which could explain the perplexing phenomenon of delayed cell death was soon supported by experimental and human data, and many researchers believe this helps explain why neural rescue works in the newborn. However the picture is complex: both apoptosis and necrosis are present in variable proportions; and there seems to be prolonged neurodegeneration after an insult. Research into this problem continues.
Neonatal neural rescue
These ideas flowed through the perinatal research community, producing a new belief that neural rescue after birth asphyxia should be possible. Amongst the first to have attempt neonatal neural rescue in animals were Ingmar Kjellmer and Henrik Hagberg in Gothenburg, and Michael Johnston in Baltimore. The potential began to draw in other neonatal researchers from diverse fields to begin neuroprotection research, including those who came to form the informal neonatal hypothermia research group:
Peter Gluckman and Tania Gunn were endocrinologists in the University of Auckland New Zealand and interested in cooling for its effect on thyroid function; they had first cooled a sheep fetus for endocrine studies in 1983. Denis Azzopardi, John Wyatt and David Edwards, then young researchers working for Reynolds, were using Reynolds’s sophisticated MRS approach to replicate secondary energy failure in newborn piglets and immature rats; in Gluckman’s laboratory Alistair Gunn and Chris Williams developed a simple and elegant biophysical method using cerebral impedance to do essentially the same thing in fetal sheep. Marianne Thoresen, who was working on cerebral perfusion, was prompted to think about neuroprotection by stories of children who fell through the Norwegian ice and suffering prolonged drowning in iced water but emerged with preserved cerebral function.
There were many potential therapies around which might achieve neural rescue, and most of these workers did not immediately move to hypothermia. Magnesium was an appealingly simple excitoxin receptor antagonist that protected cells in culture: the Reynolds group tested it in their piglet model without success. Gluckman and Gunn started by looking unsuccessfully at flunarizine, a calcium entry inhibitor. Edwards picked on nitric oxide synthase inhibition which was also a failure. Gluckman had success with his innovative studies of IGF-1, but could not immediately translate this to clinical practice.
- Jacobs, SE; Berg, M; Hunt, R; Tarnow-Mordi, WO; Inder, TE; Davis, PG (31 January 2013). "Cooling for newborns with hypoxic ischaemic encephalopathy.". The Cochrane database of systematic reviews. 1: CD003311. doi:10.1002/14651858.CD003311.pub3. PMID 23440789.
- Shankaran, S; Pappas, A; McDonald, SA; Vohr, SR; Hintz, SR; Yolton, K; Gustafson, KE; Leach, TM; Green, C; et al. (2012). "Childhood outcomes after hypothermia for neonatal encephalopathy". New England Journal of Medicine. 366 (22): 2085–92. doi:10.1056/NEJMoa1112066. PMC . PMID 22646631.
- Guillet, R; Edwards, AD; Thoresen, M; CoolCap Trial Group (2011). "Seven- to eight-year follow-up of the CoolCap trial of head cooling for neonatal encephalopathy.". Pediatr Res. 71 (2): 205–9. doi:10.1038/pr.2011.30. PMID 22258133.
- Rutherford, M; Ramenghi, LA; Edwards, AD; Brocklehurst, P; Halliday, H; Levene, M; Strohm, B; Thoresen, M; et al. (2010). "Assessment of brain tissue injury after moderate hypothermia in neonates with hypoxic-ischaemic encephalopathy: a nested substudy of a randomised controlled trial". Lancet neurology. 9 (1): 39–45. doi:10.1016/S1474-4422(09)70295-9. PMC . PMID 19896902.
- Robertson, NJ; Nakakeeto, M; Hagmann, C; Cowan, FM; Acolet, D; Iwata, O; Allen, E; Elbourne, D; et al. (2008). "Therapeutic hypothermia for birth asphyxia in low-resource settings: a pilot randomised controlled trial". Lancet. 372 (9641): 801–3. doi:10.1016/S0140-6736(08)61329-X. PMID 18774411.
- Siesjo, BK; Katsura, K; Kristian, T. (1995). "The biochemical basis of cerebral ischemic damage". Journal of Neurosurgical Anesthesiology. 7 (1): 47–52. doi:10.1097/00008506-199501000-00009. PMID 7881240.
- Siesjö, BK (1981). "Cell damage in the brain: a speculative synthesis". Journal of Cerebral Blood Flow and Metabolism. 1 (2): 155–85. doi:10.1038/jcbfm.1981.18. PMID 6276420.
- Taylor, DL; Edwards, AD; Mehmet, H. (1999). "Oxidative metabolism, apoptosis and perinatal brain injury". Brain Pathol. 9 (1): 93–117. doi:10.1111/j.1750-3639.1999.tb00213.x. PMID 9989454.
- Roth, Simon C.; Edwards, A. David; Cady, Ernest B.; Delpy, David T.; Wyatt, John S.; Azzopardi, Denis; Baudin, Jenny; Townsend, Jan; et al. (2008). "RELATION BETWEEN CEREBRAL OXIDATIVE METABOLISM FOLLOWING BIRTH ASPHYXIA, AND NEURODEVELOPMENTAL OUTCOME AND BRAIN GROWTH AT ONE YEAR". Developmental Medicine & Child Neurology. 34 (4): 285–95. doi:10.1111/j.1469-8749.1992.tb11432.x.
- Robertson, NJ; Cox, IJ; Cowan, FM; Counsell, SJ; Azzopardi, D; Edwards, AD. (1999). "Cerebral intracellular lactic alkalosis persisting months after neonatal encephalopathy measured by magnetic resonance spectroscopy". Pediatr. Res. 46 (3): 287–96. doi:10.1203/00006450-199909000-00007. PMID 10473043.
- Siesjö, BK; Elmér, E; Janelidze, S; Keep, M; Kristián, T; Ouyang, YB; Uchino, H (1999). "Role and mechanisms of secondary mitochondrial failure". Acta neurochirurgica. Supplement. 73: 7–13. PMID 10494335.
- Northington, FJ; Ferriero, DM; Graham, EM; Traystman, RJ; Martin, LJ. (2001). "Early Neurodegeneration after Hypoxia-Ischemia in Neonatal Rat Is Necrosis while Delayed Neuronal Death Is Apoptosis". Neurobiol. Dis. 8 (2): 207–19. doi:10.1006/nbdi.2000.0371. PMID 11300718.
- McDonald, J; Johnston, MV (1990). "Physiological and pathophysiological roles of excitatory amino acids during central nervous system development". Brain Research Reviews. 15 (1): 41–70. doi:10.1016/0165-0173(90)90011-C. PMID 2163714.
- Wang, X.; Carlsson, Y.; Basso, E.; Zhu, C.; Rousset, C. I.; Rasola, A.; Johansson, B. R.; Blomgren, K.; et al. (2009). "Developmental Shift of Cyclophilin D Contribution to Hypoxic-Ischemic Brain Injury". Journal of Neuroscience. 29 (8): 2588–96. doi:10.1523/JNEUROSCI.5832-08.2009. PMC . PMID 19244535.
- Northington, FJ; Ferriero, DM; Flock, DL; Martin, LJ (2001). "Delayed neurodegeneration in neonatal rat thalamus after hypoxia-ischemia is apoptosis". Journal of Neuroscience. 21 (6): 1931–8. PMID 11245678.
- Gill, Ramanjit; Soriano, Marc; Blomgren, Klas; Hagberg, Henrik; Wybrecht, Remy; Miss, Marie-Therese; Hoefer, Sandra; Adam, Geo; Niederhauser, Olivier; Kemp, John A.; Loetscher, Hansruedi (2002). "Role of Caspase-3 Activation in Cerebral Ischemia-Induced Neurodegeneration in Adult and Neonatal Brain". Journal of Cerebral Blood Flow & Metabolism: 420–430. doi:10.1097/00004647-200204000-00006.
- Bona, E; Andersson, AL; Blomgren, K; Gilland, E; Puka-Sundvall, M; Gustafson, K; Hagberg, H (1999). "Chemokine and inflammatory cell response to hypoxia-ischemia in immature rats". Pediatric research. 45 (4 Pt 1): 500–9. doi:10.1203/00006450-199904010-00008. PMID 10203141.
- Hedtjärn, M; Leverin, AL; Eriksson, K; Blomgren, K; Mallard, C; Hagberg, H (2002). "Interleukin-18 involvement in hypoxic-ischemic brain injury". Journal of Neuroscience. 22 (14): 5910–9. PMID 12122053.
- Boutin, H; Lefeuvre, RA; Horai, R; Asano, M; Iwakura, Y; Rothwell, NJ (2001). "Role of IL-1alpha and IL-1beta in ischemic brain damage". Journal of Neuroscience. 21 (15): 5528–34. PMID 11466424.
- Hedtjärn, Maj; Mallard, Carina; Iwakura, Yoichiro; Hagberg, Henrik (2005). "Combined Deficiency of IL-1β18, but Not IL-1αβ, Reduces Susceptibility to Hypoxia-Ischemia in the Immature Brain". Developmental Neuroscience. 27 (2–4): 143–8. doi:10.1159/000085986. PMID 16046848.
- Ferriero, DM (2004). "Neonatal brain injury". The New England Journal of Medicine. 351 (19): 1985–95. doi:10.1056/NEJMra041996. PMID 15525724.
- Erecinska, M; Thoresen, M; Silver, IA. (2003). "Effects of hypothermia on energy metabolism in mammalian central nervous system". J Cereb. Blood Flow Metab. 23 (5): 513–30. doi:10.1097/01.WCB.0000066287.21705.21. PMID 12771566.
- Lorek, A; Takei, Y; Cady, EB; Wyatt, JS; Penrice, J; Edwards, AD; Peebles, D; Wylezinska, M; et al. (1994). "Delayed ('secondary') cerebral energy failure following acute hypoxia-ischaemia in the newborn piglet: continuous 48-hour studies by 31P magnetic resonance spectroscopy". Pediatr Res. 36 (6): 699–706. doi:10.1203/00006450-199412000-00003. PMID 7898977.
- Amess, PN; Penrice, J; Cady, EB; Lorek, A; Wylezinska, M; Cooper, CE; D'souza, P; Tyszczuk, L; et al. (1997). "Mild hypothermia after severe transient hypoxia-ischemia reduces the delayed rise in cerebral lactate in the newborn piglet". Pediatric research. 41 (6): 803–8. doi:10.1203/00006450-199706000-00002. PMID 9167192.
- Gunn, AJ; Gunn, TR; Gunning, MI; Williams, CE; Gluckman, PD. (1998). "Neuroprotection with prolonged head cooling started before postischemic seizures in fetal sheep". Pediatrics. 102 (5): 1098–106. doi:10.1542/peds.102.5.1098. PMID 9794940.
- Smith, SL; Hall, ED. (1996). "Mild pre- and posttraumatic hypothermia attenuates blood–brain barrier damage following controlled cortical impact injury in the rat". J. Neurotrauma. 13 (1): 1–9. doi:10.1089/neu.1996.13.1. PMID 8714857.
- Busto, R; Globus, MY; Dietrich, WD; Martinez, E; Valdes, I; Ginsberg, MD. (1989). "Effect of mild hypothermia on ischemia-induced release of neurotransmitters and free fatty acids in rat brain". Stroke. 20 (7): 904–10. doi:10.1161/01.str.20.7.904. PMID 2568705.
- Thoresen, M; Satas, S; Puka-Sundvall, M; Whitelaw, A; Hallestrom, A; Loberg, E; Ungerstedt, U; Steen, PA; Hagberg, H (1997). "Post-hypoxic hypothermia reduces cerebrocortical release of NO and excitotoxins". NeuroReport. 8 (15): 3359–62. doi:10.1097/00001756-199710200-00033. PMID 9351672.
- Nakashima, K; Todd, MM. (1996). "Effects of hypothermia on the rate of excitatory amino acid release after ischemic depolarization". Stroke. 27 (5): 913–8. doi:10.1161/01.str.27.5.913. PMID 8623113.
- Globus, MY; Alonso, O; Dietrich, WD; Busto, R; Ginsberg, MD. (1995). "Glutamate release and free radical production following brain injury: effects of posttraumatic hypothermia". J. Neurochem. 65 (4): 1704–11. doi:10.1046/j.1471-4159.1995.65041704.x. PMID 7561868.
- Akisu, M; Huseyinov, A; Yalaz, M; Cetin, H; Kultursay, N. (2003). "Selective head cooling with hypothermia suppresses the generation of platelet-activating factor in cerebrospinal fluid of newborn infants with perinatal asphyxia". Prostaglandins Leukot. Essent. Fatty Acids. 69 (1): 45–50. doi:10.1016/S0952-3278(03)00055-3. PMID 12878450.
- Edwards, AD; Yue, X; Squier, MV; Thoresen, M; Cady, EB; Penrice, J; Cooper, CE; Wyatt, JS; et al. (1995). "Specific inhibition of apoptosis after cerebral hypoxia-ischaemia by moderate post-insult hypothermia". Biochem. Biophys. Res. Commun. 217 (3): 1193–9. doi:10.1006/bbrc.1995.2895. PMID 8554576.
- Xu, L; Yenari, MA; Steinberg, GK; Giffard, RG. (2002). "Mild hypothermia reduces apoptosis of mouse neurons in vitro early in the cascade". J Cereb. Blood Flow Metab. 22 (1): 21–8. doi:10.1097/00004647-200201000-00003. PMID 11807390.
- Zhang, Z; Sobel, RA; Cheng, D; Steinberg, GK; Yenari, MA. (2001). "Mild hypothermia increases Bcl-2 protein expression following global cerebral ischemia. Brain Res. Mol". Brain Res. 95: 75–85. doi:10.1016/S0169-328X(01)00247-9. PMID 11687278.
- Wang, H; Olivero, W; Wang, D; Lanzino, G (2006). "Cold as a therapeutic agent". Acta neurochirurgica. 148 (5): 565–570. doi:10.1007/s00701-006-0747-z. PMID 16489500.
- Little, WJ. (1966). "On the influence of abnormal parturition, difficult labours, premature birth, and asphyxia neonatorum, on the mental and physical condition of the child, especially in relation to deformities". Clin. Orthop. Relat Res. 46: 7–22. doi:10.1097/00003086-196600460-00002. PMID 5950310.
- Westin, B. (2006). "Hypothermia in the resuscitation of the neonate: a glance in my rear-view mirror". Acta Paediatr. 95 (10): 1172–4. doi:10.1080/08035250600794583. PMID 16982485.
- Miller, JA (1949). "Temperature and Survival of Newborn Guinea Pigs Under Anoxia". Science. 110 (2848): 113–4. doi:10.1126/science.110.2848.113. PMID 17780238.
- Enhorning, G; Westin, B. (1954). "Experimental studies of the human fetus in prolonged asphyxia". Acta Physiol Scand. 31 (4): 359–75. doi:10.1111/j.1748-1716.1954.tb01147.x. PMID 13197106.
- Westin, B; Miller, JA; Nyberg, R; Wedenberg, E. (1959). "Neonatal asphyxia pallida treated with hypothermia alone or with hypothermia and transfusion of oxygenated blood". Surgery. 45 (5): 868–79. PMID 13659328.
- Auld, PA; Nelson, NM; Nicopopoulos, DA; Helwig, F; Smith, CA. (1962). "Physiologic studies on an infant in deep hypothermia". N. Engl. J. Med. 267 (26): 1348–51. doi:10.1056/NEJM196212272672606. PMID 13965545.
- Silverman, WS; Fertig, JW; Berger, AP. (1958). "The influence of the thermal environment upon the survival of newly born premature infants". Pediatrics. 22 (5): 876–85. PMID 13600915.
- Mann, TP; Elliott, RIK (1957). "Neonatal cold injury due to accidental exposure to cold". Lancet. 272 (6962): 229–34. doi:10.1016/s0140-6736(57)90298-2. PMID 13399181.
- Brodie, HR; Cross, KW; Lomer, TR. (1957). "Heat production in new-born infants under normal and hypoxic conditions". J. Physiol. 138 (1): 156–63. PMC . PMID 13463804.
- Miller, JA; Jr, R; Zakary, R; Miller, FS. (1964). "Hypothermia, Asphyxia, and cardiac glycogen in guinea pigs". Science. 144 (3623): 1226–7. doi:10.1126/science.144.3623.1226. PMID 14150326.
- Dunn, JM; Miller, JA (1969). "Hypothermia combined with positive pressure ventilation in resuscitation of the asphyxiated neonate. Clinical observations in 28 infants". Am. J. Obstet. Gynecol. 104 (1): 58–67. PMID 4888017.
- Ehrstrom, J; Hirvensalo, M; Donner, M; Hietalahti, J. (1969). "Hypothermia in the resuscitation of severely asphyctic newborn infants. A follow-up study". Ann. Clin. Res. 1 (1): 40–9. PMID 5350770.
- Cordey, R; Chiolero, R; Miller, JA; Jr (1973). "Resuscitation of neonates by hypothermia: report on 20 cases with acid-base determination on 10 cases and the long-term development of 33 cases". Resuscitation. 2 (3): 169–81. doi:10.1016/0300-9572(73)90042-7. PMID 4773063.
- Oates, RK; Harvey, D. (1976). "Failure of hypothermia as treatment for asphyxiated newborn rabbits". Arch. Dis. Child. 51 (7): 512–6. doi:10.1136/adc.51.7.512. PMC . PMID 989263.
- Michenfelder, JD; Milde, JH. (1977). "Failure of prolonged hypocapnia, hypothermia, or hypertension to favorably alter acute stroke in primates". Stroke. 8 (1): 87–91. doi:10.1161/01.str.8.1.87. PMID 402043.
- Bohn, DJ; Biggar, WD; Smith, CR; Conn, AW; Barker, GA. (1986). "Influence of hypothermia, barbiturate therapy, and intracranial pressure monitoring on morbidity and mortality after near-drowning". Crit Care Med. 14 (6): 529–34. doi:10.1097/00003246-198606000-00002. PMID 3709193.
- Kopshev, SN (1982). "Craniocerebral hypothermia in the prevention and combined therapy of cerebral pathology in infants with asphyxia neonatorum". Akusherstvo i ginekologiia (7): 56–8. PMID 7137497.
- Prof Peter Dunn, Bristol, personal communication
- Delpy, DT; Gordon, RE; Hope, PL; Parker, D; Reynolds, EO; Shaw, D; Whitehead, MD (1982). "Noninvasive investigation of cerebral ischemia by phosphorus nuclear magnetic resonance". Pediatrics. 70 (2): 310–3. PMID 7099806.
- Vannucci, RC. (1990). "Experimental biology of cerebral hypoxia-ischemia: Relation to perinatal brain damage". Pediatr. Res. 27 (4 Pt 1): 317–26. doi:10.1203/00006450-199004000-00001. PMID 1971436.
- Kirino, T. (1982). "Delayed neuronal death in the gerbil hippocampus following ischemia". Brain Res. 239 (1): 57–69. doi:10.1016/0006-8993(82)90833-2. PMID 7093691.
- Pulsinelli, WA; Brierley, JB; Plum, F. (1982). "Temporal profile of neuronal damage in a model of transient forebrain ischemia". Annals of Neurology. 11 (5): 491–8. doi:10.1002/ana.410110509. PMID 7103425.
- Hope, PL; Costello, AM; Cady, EB; Delpy, DT; Tofts, PS; Chu, A; Hamilton, PA; Reynolds, EO; Wilkie, DR (1984). "Cerebral energy metabolism studied with phosphorus NMR spectroscopy in normal and birth-asphyxiated infants". Lancet. 2 (8399): 366–70. doi:10.1016/s0140-6736(84)90539-7. PMID 6147452.
- Olney, JW; Sharpe, LG. (1969). "Brain lesions in an infant rhesus monkey treated with monsodium glutamate". Science. 166 (3903): 386–8. doi:10.1126/science.166.3903.386. PMID 5812037.
- Olney, JW; Ho, OL. (1970). "Brain damage in infant mice following oral intake of glutamate, aspartate or cysteine". Nature. 227 (5258): 609–11. doi:10.1038/227609b0. PMID 5464249.
- Simon, RP; Swan, JH; Griffiths, T; Meldrum, BS. (1984). "Blockade of N-methyl-D-aspartate receptors may protect against ischemic damage in the brain". Science. 226 (4676): 850–2. doi:10.1126/science.6093256. PMID 6093256.
- Kerr, JF; Wyllie, AH; Currie, AR. (1972). "Apoptosis, a basic biological phenomenon with wide-ranging implications in human tissue kinetics". British Journal of Cancer. 26 (4): 239–57. doi:10.1038/bjc.1972.33. PMC . PMID 4561027.
- Ellis, HM; Horvitz, HR. (1986). "Genetic control of programmed cell death in the nematode C. elegans". Cell. 44 (6): 817–29. doi:10.1016/0092-8674(86)90004-8. PMID 3955651.
- Raff, MC. (1992). "Social controls on cell survival and cell death". Nature. 356 (6368): 397–400. doi:10.1038/356397a0. PMID 1557121.
- Evan, GI; Wyllie, AH; Gilbert, CS; Littlewood, TD; Land, H; Brooks, M; Waters, CM; Penn, LZ; Hancock, DC (1992). "Induction of apoptosis in fibroblasts by c-myc protein". Cell. 69 (1): 119–28. doi:10.1016/0092-8674(92)90123-T. PMID 1555236.
- Mehmet, H; Yue, X; Squier, MV; Lorek, A; Cady, E; Penrice, J; Sarraf, C; Wylezinska, M; et al. (1994). "Increased apoptosis in the cingulate sulcus of newborn piglets following transient hypoxia-ischaemia is related to the degree of high energy phosphate depletion during the insult". Neurosci. Lett. 181 (1–2): 121–5. doi:10.1016/0304-3940(94)90574-6. PMID 7898750.
- Beilharz, E; Williams, CE; Dragunow, M; Sirimanne, E; Gluckman, PD. (1995). "Mechanisms of cell death following hypoxic-ischaemic injury in the immature rat: evidence of apoptosis during selective neuronal loss". Mol. Brain. Res. 29 (1): 1–14. doi:10.1016/0169-328X(94)00217-3. PMID 7769986.
- Edwards, AD; Yue, X; Cox, P; Hope, PL; Azzopardi, D; Squier, M. V.; Mehmet, H (1997). "Apoptosis in the brains of infants suffering intrauterine cerebral injury". Pediatr Res. 42 (5): 684–9. doi:10.1203/00006450-199711000-00022. PMID 9357944.
- Northington, FJ; Graham, EM; Martin, LJ. (2005). "Poptosis in perinatal hypoxic-ischemic brain injury: how important is it and should it be inhibited?". Brain Res Rev. 50 (2): 244–57. doi:10.1016/j.brainresrev.2005.07.003. PMID 16216332.
- Stone BS, Zhang J, Mack DW, Mori S, Martin LJ, Northington FJ (Nov 2008). "Delayed neural network degeneration after neonatal hypoxia-ischemia". Ann Neurol. 64 (5): 535–46. doi:10.1002/ana.21517.
- Thiringer, K; Hrbek, A; Karlsson, K; Rosen, KG; Kjellmer, I. (1987). "Postasphyxial cerebral survival in newborn sheep after treatment with oxygen free radical scavengers and a calcium antagonist". Pediatr. Res. 22 (1): 62–6. doi:10.1203/00006450-198707000-00015. PMID 3627874.
- Hagberg, H; Andersson, P; Kjellmer, I; Thiringer, K; Thordstein, M. (1987). "Extracellular overflow of glutamate, aspartate, GABA and taurine in the cortex and basal ganglia of fetal lambs during hypoxia-ischemia". Neurosci. Lett. 78 (3): 311–7. doi:10.1016/0304-3940(87)90379-X. PMID 2888062.
- McDonald, JW; Silverstein, FS; Johnston, MV. (1987). "MK-801 protects the neonatal brain from hypoxic-ischemic damage". Eur. J. Pharmacol. 140 (3): 359–61. doi:10.1016/0014-2999(87)90295-0. PMID 2820765.
- Blumberg, RM; Cady, EB; Wigglesworth, JS; McKenzie, JE; Edwards, AD. (1996). "Relation between delayed impairment of cerebral energy metabolism and infarction following transient focal hypoxia ischaemia in the developing brain. Exp". Brain Research. 113 (1): 130–7. doi:10.1007/BF02454148. PMID 9028781.
- Williams, CE; Gunn, AJ; Mallard, C; Gluckman, PD. (1992). "Outcome after ischemia in the developing sheep brain: an electroencephalographic and histological study". Annals of Neurology. 31 (1): 14–21. doi:10.1002/ana.410310104. PMID 1543346.
- Clemence, M; Thornton, JS; Penrice, J; Amess, P; Punwani, S; Tyszczuk, L; et al. (1996). "31P MRS and quantitative diffusion and T2 MRI show no cerebroprotective effects of intravenous MgSO4 after severe transient hypoxia-ischaemia in the neonatal piglet". MAGMA. 4: 114.
- Gunn, AJ; Mydlar, T; Bennet, L; Faull, RL; Gorter, S; Cook, C; Johnston, BM; Gluckman, PD (1989). "The neuroprotective actions of a calcium channel antagonist, flunarizine, in the infant rat". Pediatr. Res. 25 (6): 573–6. doi:10.1203/00006450-198906000-00003. PMID 2740146.
- Marks, KA; Mallard, C; Roberts, I; Williams, C; Gluckman, P; Edwards, AD. (1996). "Nitric oxide synthase inhibition attenuates delayed vasodilation and increases injury following cerebral ischaemia in fetal sheep". Pediatr Res. 40 (2): 185–91. doi:10.1203/00006450-199608000-00002. PMID 8827765.
- Gluckman, PD; Klempt, N; Guan, J; Mallard, C; Sirimanne, E; Dragunow, M; Klempt, M; Singh, K; et al. (1992). "A role for IGF-1 in the rescue of CNS neurons following hypoxic- ischemic injury". Biochem. Biophys. Res. Commun. 182 (2): 593–9. doi:10.1016/0006-291X(92)91774-K. PMID 1370886.