THERAPEUTIC HYPOTHERMIA – MECHANISMS OF ACTION

Therapeutic hypothermia

– mechanisms of action

D-6695-2018
Dr A W Niranjan Thomas
Professor and Head Unit 1, Department of Neonatology, Christian Medical College, Vellore

Dr Niranjan Thomas is certified neonatologist, he completed his fellowship degree in neonatal and perinatal
medicine from the Hospital of Sick Children, University of Toronto in Canada. Dr. Thomas’ research is focused
mainly around neuroprotection in HIE and Neonatal nutrition. Dr. Thomas has been awarded with Best
innovation Poster award NEOCON 2012 for his research “A low cost effective method to deliver therapeutic
hypothermia for babies with HIE using phase changing material”. Dr. Niranjan also played a key role as a clinical
consultant in the Development of a low cost solution for providing therapeutic hypothermia for babies with HIE
based on phase changing material, and he has also been granted a patent for this innovative product.

© Drägerwerk AG & Co. KGaA 1


Introduction
Perinatal asphyxia is a leading cause of neonatal mortality, accounting
for 23% of the 3.6 million neonatal deaths worldwide (1). Hypoxic
ischemic encephalopathy (HIE) occurs in 1–2 per 1,000 live births
in high-income countries (2). In low- and middle-income countries
(LMICs) like India, the incidence is 14 per 1,000 live births (3).
According to the recent Cochrane meta-analysis, approximately 35%
of infants with moderate to severe HIE die and 25% develop major
neuro-developmental disability (4).
Mechanism of neuronal injury in HIE
There are 2 phases of neuronal injury following perinatal asphyxia -
primary and secondary energy failure (5,6). Primary energy failure
starts immediately after the asphyxia insult (Figure 1). Hypoxia and
ischemia lead to anaerobic metabolism and ATP depletion, leading
to loss of ionic homeostasis and neuronal cell death. There is also
excitotoxic and free radical mediated injury during this phase.
Figure 1: The Biphasic Pattern of Energy Failure in Neuronal
Injury Following Perinatal Asphyxia [Adapted from: Robertson NJ,
Groenendaal F. Hypoxic Ischemic Brain Injury. In. Rennie JM, editor.
Rennie and Roberton’s Textbook of Neonatology. 5th Ed. Churchill
Livingstone; 2012.p.1102-55.]
Primary energy failure is followed by a latent phase, during
which many cells recover at least partially from the primary
insult and oxidative metabolism is partially restored following re-
establishment of cerebral blood flow.
The phase of secondary energy failure occurs 6–15 hours after
the initial insult, reaches its nadir by 24-48 hours and may
THERAPEUTIC HYPOTHERMIA – MECHANISMS OF ACTION
continue for days to weeks. During this phase, a second cascade
of neuronal injury occurs as a result of multiple mechanisms such
as accumulation of excitatory neurotransmitters (particularly
glutamate), increase in cytosolic calcium, activation of inflammation
and generation of free radicals.
Therapeutic window
The latent phase is the therapeutic window. The goal is to initiate
therapy before secondary energy failure sets in. In experimental
animal models, the onset of secondary energy failure and
seizures occurs approximately 6 hours after the insult. Initiation
of therapies including TH during the therapeutic window has
been successful in reducing brain damage.
Therapeutic Hypothermia
To date, TH is the only promising neuroprotective intervention in
HIE and has become the standard of care in developed countries
(7,8). It is carried out for neonates with strict inclusion criteria
and with a standard protocol (9–12). The target temperature
during TH is 33.5±0.5º C, though it varies slightly between
trials. TH is initiated within 6 hours after birth and continued
for 72 hours, followed by slow rewarming over 10-12 hours
(Figure 2).
Figure 2: Protocol for Therapeutic Hypothermia in Asphyxiated Neonates
[Adapted from: Robertson NJ, Groenendaal F. Hypoxic Ischemic
Brain Injury. In. Rennie JM, editor. Rennie and Roberton’s Textbook of
Neonatology. 5th Ed. Churchill Livingstone; 2012.p.1102-55.]
© Drägerwerk AG & Co. KGaA 2

TH reduces mortality by 25% (number need to treat for an additional
beneficial outcome (NNTB) 7) and major neurodevelopmental
disability at 18-24 months by 23% (NNTB 8) in infants with moderate
to severe HIE (4). TH is also shown to improve survival and cognitive
outcomes in later childhood (13,14).
Mechanism of Neuroprotection of TH in HIE
The beneficial effects of TH occur at multiple sites in the cascade
of neuronal injury leading to cell death (Figure 3) (5,6). TH causes
a decrease in energy consumption, decrease in the accumulation of
extracellular glutamate, decrease in the generation of reactive oxygen
(ROS) and nitrogen species (RNS), inhibition of inflammatory
mechanisms and interruption of downstream molecular cascades
to apoptosis. The relative importance of each effect remains to be
clarified (6).
Figure 3: Flow diagram depicting the mechanisms of
neuroprotection of Therapeutic Hypothermia in HIE
Reduction in energy consumption
The major mechanism of neuronal death in the primary phase
is depletion of ATP. Hypoxia and ischemia lead to anaerobic
metabolism and ATP depletion, resulting in failure of Na/K
ATPase membrane pump and loss of ionic homeostasis. If the
THERAPEUTIC HYPOTHERMIA – MECHANISMS OF ACTION
insult is severe, an influx of ions and water causes early cell death
by necrosis. In the more typical, less severe insult, membrane
depolarization occurs and is followed by extracellular accumulation
of glutamate, increased cytosolic calcium and a cascade of events
leading to delayed cell death by apoptosis. Hypothermia causes
a graded reduction in cerebral metabolism of about 5% for every
degree of temperature reduction (15), which delays the onset of
anoxic cell depolarization.
TH also attenuates the other two mechanisms of injury
during the primary phase; TH reduces the accumulation of
extracellular glutamate, primarily by the delay in depolarization,
and suppresses nitric oxide (NO) and superoxide formation,
presumptively caused by slowing of chemical reactions (6).
However, since TH is never commenced during the primary
energy failure phase, these mechanisms of neuroprotection are
less important.
Attenuation of excitotoxicity
Glutamate is the major mediator of excitotoxic injury. An increase
in extracellular glutamate results from; a) excessive presynaptic
glutamate release due to membrane depolarization and increased
cytosolic calcium and b) failure of glutamate uptake mechanisms
in presynaptic nerve endings and astrocytes due to membrane
depolarization and failure of glutamate transporters (5). TH is
beneficial both by inhibiting glutamate release and by ameliorating
the disturbance of glutamate transporters (6).
Reduction in the accumulation of
intracellular calcium
The increase in cytosolic calcium is a consequence of failure
of energy-dependent calcium-pumping mechanisms due to
depletion of ATP, opening of voltage-dependent calcium channels
secondary to membrane depolarization and activation of NMDA
and AMPA glutamate receptors (5). The deleterious effects of
increased cytosolic calcium include degradation of cellular lipids
proteins and DNA by activation of phospholipases, proteases and
nucleases respectively and indirect mechanisms of destruction
mediated by the generation of free radicals and nitricoxide. TH
attenuates intracellular accumulation of calcium by decreasing
ATP depletion, preventing membrane depolarization and reducing
glutamate mediated excitotoxicity (5).
© Drägerwerk AG & Co. KGaA 3
 THERAPEUTIC HYPOTHERMIA – MECHANISMS OF ACTION

Reduction of free radicals mediated damage

The major free radicals in mammalian cells include ROS, such
as superoxide anion, hydrogen peroxide and hydroxyl radical and
RNS, such as peroxynitrite (ONOO−). They are generated from
adenosine, NO and arachidonic acid synthesis pathways. They
react with and damage membrane phospholipids and nuclear
components resulting in cell death. TH inhibits NO synthesis and
decreases the production of other free radicals (5,6).
Conclusion
TH is the only promising intervention to improve survival
and neurodevelopmental outcomes in newborn infants with
HIE. The neuroprotective effects of TH occur by multiple
mechanisms, which ameliorate almost every step in the
cascade of injury that leads to neuronal cell death.

Blockade of apoptotic pathways

Neuronal cell death in HIE occurs by four different mechanisms: s
apoptosis, necrosis, necroptosis and autophagy (5). Though TH is
protective against all forms of cell death, it is especially protective
against apoptosis.

In HIE, apoptosis is activated by a) excessive calcium influx that

promotes depolarization and permeabilization of the mitochondrial
membrane, resulting in release of proapoptotic proteins including
cytochrome c (intrinsic pathway) and b) intense activation
of various caspases particularly caspase-3 by inflammatory
mediators and free radicals (extrinsic pathway). TH reduces
cytochrome c translocation and blocks the activation of caspases,
thus suppressing both the intrinsic and extrinsic pathways of
apoptosis (5,6).

Anti-inflammatory effects
Hypoxia-ischemia initiates a deleterious cascade of inflammatory
reactions that contributes to neuronal injury. The inflammatory
cascade is initiated by activation of microglia, which is followed by
the accumulation of neutrophils and mononuclear phagocytic cells,
release of cytokines, generation of ROS and RNS and accentuation
of excitotoxicity (5). TH prevents the inflammatory reactions mainly
by suppressing microglial activation (5,6). It also attenuates
accumulation of inflammatory cells and release of cytokines.

Other putative mechanisms

TH attenuates neuronal injury by preventing the breakdown
of blood brain barrier, decreasing cytotoxic cerebral edema and
decreasing the propensity to seizures (6). Moreover, TH is shown
to promote recovery and repair by stimulating the production of
growth factors and activation of endogenous stem cells (6).

© Drägerwerk AG & Co. KGaA 4

 THERAPEUTIC HYPOTHERMIA – MECHANISMS OF ACTION

REFERENCE:

1. Lawn JE, Kerber K, Enweronu-Laryea C, Cousens S. 3.6 million neonatal deaths--what is progressing and what is not? SeminPerinatol. 2010 Dec;34(6):371–86.

2. Kurinczuk JJ, White-Koning M, Badawi N. Epidemiology of neonatal encephalopathy and hypoxic-ischaemic encephalopathy. Early Hum Dev. 2010 Jun;86(6):329–38.

3. National Perinatal Neonatal Database. Available from URL: www.newbornwhocc.org/pdf/nnpd_report_2002-03.PDF: Accessed Apr 11, 2016.

4. Jacobs SE, Berg M, Hunt R, Tarnow-Mordi WO, Inder TE, Davis PG. Cooling for newborns with hypoxic ischaemic encephalopathy. Cochrane Database Syst Rev. 2013;1:CD003311.

5. Inder TE, Volpe JJ. Pathophysiology: General Principles. In: Volpe JJ, Inder TE, Darras BT, de Vries LS, du Plessis AJ, Neil JJ, Perlman JM, editors. Volpe’s Neurology of the Newborn.
6th ed. Philadelphia: Elsevier; 2018.p.325-88. In.

6. Drury PP, Gunn ER, Bennet L, Gunn AJ. Mechanisms of hypothermic neuroprotection. Clin Perinatol. 2014 Mar;41(1):161–75.

7. Shankaran S. Hypoxic-ischemic encephalopathy and novel strategies for neuroprotection. Clin Perinatol. 2012 Dec;39(4):919–29.

8. Kapetanakis A, Azzopardi D, Wyatt J, Robertson NJ. Therapeutic hypothermia for neonatal encephalopathy: a UK survey of opinion, practice and neuro-investigation at the end of
2007. Acta Paediatr Oslo Nor 1992. 2009 Apr;98(4):631–5.

9. Shankaran S, Laptook AR, Ehrenkranz RA, Tyson JE, McDonald SA, Donovan EF, et al. Whole-body hypothermia for neonates with hypoxic-ischemic encephalopathy. N Engl J
Med. 2005 Oct 13;353(15):1574–84.

10. Azzopardi DV, Strohm B, Edwards AD, Dyet L, Halliday HL, Juszczak E, et al. Moderate hypothermia to treat perinatal asphyxial encephalopathy. N Engl J Med.
2009;361(14):1349–58.

11. Jacobs SE. Whole-Body Hypothermia for Term and Near-Term Newborns With Hypoxic-Ischemic Encephalopathy: A Randomized Controlled Trial. Arch PediatrAdolesc Med. 2011
Aug 1;165(8):692.

12. Thomas N, Abiramalatha T, Bhat V, Varanattu M, Rao S, Wazir S, et al. Phase Changing Material for Therapeutic Hypothermia in Neonates with Hypoxic Ischemic Encephalopa-
thy - A Multi-centric Study. Indian Pediatr. 2018 15;55(3):201–5.

13. Shankaran S, Pappas A, McDonald SA, Vohr BR, Hintz SR, Yolton K, et al. Childhood outcomes after hypothermia for neonatal encephalopathy. N Engl J Med. 2012 May
31;366(22):2085–92.

14. Azzopardi D, Strohm B, Marlow N, Brocklehurst P, Deierl A, Eddama O, et al. Effects of hypothermia for perinatal asphyxia on childhood outcomes. N Engl J Med. 2014 Jul
10;371(2):140–9.

15. Laptook AR, Corbett RJ, Sterett R, Garcia D, Tollefsbol G. Quantitative relationship between brain temperature and energy utilization rate measured in vivo using 31P and 1H
magnetic resonance spectroscopy. Pediatr Res. 1995 Dec;38(6):919–25.

IMPRINT
SINGAPORE
Regional office for AAA Region
(Asia, Australia, Africa)
Draeger Singapore Pte Ltd
25 International Business Park #04-20
Singapore 609916
PDF 9018

www.draeger.com

© Drägerwerk AG & Co. KGaA 5