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Pharmacologic Neuroprotective
Strategies in Neonatal Brain
Injury
Sandra E. Juul, MD, PhD
a,
*, Donna M. Ferriero, MD, MS
b
MECHANISMS OF BRAIN INJURY: PRETERM VERSUS TERM
The two most common causes of neonatal brain injury in the United States are
extreme prematurity and hypoxic-ischemic encephalopathy (HIE). In the United
States, 1 in 8 babies is born before term (37–40 weeks), and 1.44% of babies
(56,000 per year) are born with a birth weight of 1250 g or less.
1
These small, preterm
babies are at high risk of death or neurodevelopmental impairment: approximately
20% die before hospital discharge, and 40% of survivors develop long-term intellec-
tual or physical impairment, including cerebral palsy (CP).
2–4
Care of preterm infants
accounts for more than half of pediatric health care dollars spent.
The brain rapidly increases in size, shape, and complexity during the second and
third trimesters.
5
Neurodevelopmental compromise can result from an interruption
of normal development or from damage to existing tissues. Brain development during
this period is vulnerable to hypoxia-ischemia (HI), oxidant stress, inflammation, exci-
totoxicity, and poor nutrition. These exposures can result in structural, biochemical,
Disclosure Statement: Neither author has anything to disclose.
a
Department of Pediatrics, University of Washington, 1959 Northeast Pacific Street,
Box 356320, Seattle, WA 98195, USA;
b
Neonatal Brain Disorders Laboratory, University of
California, San Francisco, 675 Nelson Rising Lane, Room 494, Box 0663, San Francisco, CA
94143, USA
* Corresponding author.
E-mail address: sjuul@uw.edu
KEYWORDS
Brain injury Hypoxic-ischemic encephalopathy Prematurity Preconditioning
KEY POINTS
There are many ways to achieve neuroprotection: preconditioning, salvaging, repair.
Hypothermia is now standard of care for term hypoxic-ischemic encephalopathy so
studies to investigate additional therapies will be added to that treatment.
Strategies that target multiple mechanisms and consider age-appropriate mechanisms
will be most beneficial.
Clin Perinatol 41 (2014) 119–131
http://dx.doi.org/10.1016/j.clp.2013.09.004 perinatology.theclinics.com
0095-5108/14/$ – see front matter Published by Elsevier Inc.
and cell-specific injury.
6
Preoligodendrocytes, which emerge and mature between 24
and 32 weeks of development, are particularly susceptible to injury, and damage to
these cells can result in white matter injury.
7
Although intracranial hemorrhage, peri-
ventricular leukomalacia, inflammatory conditions, and male gender are known risk
factors for poor outcomes, little is known about how to improve these outcomes.
HIE is estimated to contribute significantly to 23% of the 4 million neonatal deaths
that occur annually.
8
In the United States, HIE occurs in 1.5 to 2 live births per 1000,
with a higher incidence in premature infants.
9
Untreated, the sequelae of moderate to
severe HIE includes a 60% to 65% risk of mental retardation, CP, hydrocephalus, sei-
zures, or death. Perinatal inflammation is increasingly recognized as an important
contributor to neonatal HIE and poor neurodevelopmental outcomes
10
: the presence
of maternal fever alone increases the risk for CP, and chorioamnionitis further in-
creases the risks for brain injury in both preterm and term infants.
11,12
Timing of infec-
tion/inflammation relative to hypoxia is critical: it can be sensitizing (increase brain
injury) if it occurs acutely or after 72 hours, but may be protective if it occurs 24 hours
before hypoxia.
13
This differential response is not fully understood, but may depend on
activation of fetal/neonatal Toll-like receptors in the brain.
14,15
Understanding the
complex mechanisms of brain injury is essential to devising protective strategies.
THE INJURY CASCADE
Although the cellular targets of HI are different depending on age and severity of
insult, the basic cascade of injury occurs in a uniform way regardless of age and con-
tinues for a prolonged period of time. Cell death occurs in 2 main phases: primary
death from hypoxia and energy depletion, followed by reperfusion and increased
free radical (FR) formation, excitotoxicity, and nitric oxide production with secondary
energy failure and delayed death (Fig. 1). A tertiary phase was recently proposed, in
which factors can worsen outcome, predispose a newborn to further injury, or prevent
repair or regeneration after an initial insult to the brain.
16
Such mechanisms include
persistent inflammation and epigenetic changes, which cause a blockade of oligo-
dendrocyte maturation, impaired neurogenesis, impaired axonal growth, or altered
synaptogenesis.
The injury process begins with energy failure creating excitotoxicity, which is
caused by excessive glutamatergic activation that leads to progression of HI brain
injury. Glutamate plays a key role in development, affecting progenitor cell prolifera-
tion, differentiation, migration, and survival. Glutamate accumulates in the brain after
Fig. 1. The injury cascade as it occurs over time. Potential therapeutics are inserted during
the course of the cascade. See text for details on these agents. Epo, erythropoietin.
Juul & Ferriero
120
HI
17
from a variety of causes, including vesicular release from axons and reversal
of glutamate transporters. Glutamatergic receptors include N-methyl-D-aspartate
(NMDA), alpha-3-amino-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), and
kainate. Developmental differences in glutamate receptor expression contribute to
the vulnerability of the immature brain (reviewed in Ref.
18
). NMDA receptor activation,
although important for synaptic plasticity and synaptogenesis, can increase intracel-
lular calcium, proapoptotic pathways via caspase-3 activation, FR formation, and
lipid peroxidation, resulting in profound and widespread injury to the developing
brain.
Oxidative stress is an important component of early injury, along with excitotoxicity,
to the neonatal brain resulting from the excess formation of FRs (reactive oxygen spe-
cies and reactive nitrogen species) under pathologic conditions. These FRs include
superoxide anion (O
2
), hydroxyl radical (OH), singlet oxygen (
1
O
2
), and hydrogen
peroxide (H
2
O
2
). FRs target lipids, protein, and DNA, causing damage to these cellular
components and initiating a cascade that results in cell death.
19
These deleterious biological events trigger inflammatory processes that initially are
harmful and later may be beneficial to the repair processes that occur after injury. The
inflammatory response and cytokine production that accompany infection may play a
large role in cell damage and loss. Local microglia are activated early and produce
proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha, interleukin
(IL)-1b, and IL-6, as well as glutamate, FRs, and nitric oxide (NO), and are the main
immunocompetent cells in the immature brain. Depending on the stimulus, molecular
context, and timing, these cells acquire various phenotypes, which are critical to the
outcome of the injury.
20
Cell death occurs throughout the cascade, moving from a purely necrotic type to
apoptosis with a continuum of phenotypes emerging in the developing brain (the
apoptosis-necrosis continuum). Throughout this process, mechanistic interactions
between cell death and hybrid forms of cell death occur, such as programmed or regu-
lated necrosis or necroptosis.
21
The mechanisms behind programmed necrosis in
neonatal brain injury are still being investigated but are regulated by the inflammatory
processes, especially the TNF receptor superfamily, which is activated early in injury.
There are many pharmacologic agents that can affect these injury phases (Table 1).
This article highlights some of the important future therapeutics.
Table 1
Best pharmacologic candidates for impact on injury phases
Best Candidates
Antenatal Postnatal
BH4 Melatonin
Melatonin Epo
nNOS inhibitors NAC
Xenon Xenon
Allopurinol Allopurinol
Vitamin C and E Vitamin C and E
Resveratrol Resveratrol
NAC Memantine
— Topiramate
Abbreviations: BH4, tetrahydrobiopterin; NAC, N-acetyl-L-cysteine; nNOS, neuronal nitric oxide
synthase.
Strategies in Neonatal Brain Injury 121
TARGETING THE INJURY RESPONSE
Antiexcitotoxic Agents
The earliest pharmacologic strategies to protect the newborn brain were designed to
block the initial phases of injury, excitotoxicity, and oxidative stress. Many of these
agents failed because it is impossible to block normal developmental processes,
like glutamatergic signaling, without harming the brain. Therefore, therapies designed
to block the NMDA receptor resulted in increased, rather than decreased, cell death.
22
However, some agents, like magnesium sulfate, used to stop preterm labor, seemed
to have beneficial effects even though they blocked the NMDA receptor. Recent clin-
ical trials have supported the use of antenatal magnesium.
23
In 16 hospitals in
Australia and New Zealand, 1062 women with fetuses younger than 30 weeks’ gesta-
tion were given a loading infusion of 16 mmol followed by 8 mmol MgSO
4
for up to
24 hours. Substantial gross motor dysfunction (3.4% vs 6.6%; relative risk (RR),
0.51; 95% confidence interval [CI], 0.29–0.91) and combined death or substantial
gross motor dysfunction were significantly reduced in the MgSO
4
group, although
there were no significant differences in mortality or CP in survivors. In the United
States, antenatal MgSO
4
did not reduce the risk of the composite outcome of CP or
death, much like the Australian study, but was seen to reduce moderate to severe
CP without increasing the risk of death.
23
However, the number needed to treat
was high at 56 (95% CI, 34–164).
Another potential antiexcitotoxic agent is xenon, a noble gas used as an anesthetic
agent. It has action against the NMDA receptor and has been shown to be an effective
agent against hypoxic-ischemic insult both to cortical neurons in vitro and in several
in vivo models.
24
Xenon lacks the dopamine-releasing properties that are present in
other NMDA antagonists and does not cause increased apoptotic cell death as
seen with other NMDA antagonists. Perhaps the most promising aspect of xenon
pharmacology is that it increases the translational efficiency of hypoxia inducible
factor-1 (HIF-1) through a mammalian target of rapamycin (mTOR) pathway
25
that
has resulted in its potential application in HIE for both postinjury treatment and as a
preconditioner. The prolonged increase in expression of HIF-1a by xenon causes
upregulation of cytoprotective proteins such as erythropoietin (Epo), vascular endo-
thelial growth factor (VEGF), and glucose transporter 1 protein.
25
When given with
sevoflurane during labor to rats, it preconditions the fetal brain against a subsequent
HI insult.
26
When given to neonatal rats in combination with hypothermia, it improves
both functional and structural outcomes, even when hypothermia is delayed, and the
effect is sustained through adulthood.
27
Antioxidants
Therapies designed to reduce oxidative stress have proved to be efficacious both in
the preterm and term injury states. Allopurinol was originally shown to be neuroprotec-
tive in postnatal day 7 rats after HI
28
but in humans was not seen to improve short-term
or long-term outcomes in a small trial after birth asphyxia.
29
It was postulated that the
drug needed to be given before reperfusion injury set in, so trials are now underway to
evaluate efficacy when given to mothers who have fetuses suspected of intrauterine
hypoxia. In a randomized, double-blind, placebo-controlled multicenter study that is
now in progress, intravenous allopurinol is being given antenatally with the primary
outcome being serum brain damage markers (S100b) and oxidative stress markers
(isoprostanes and so forth) in umbilical cord blood; secondary outcome measures
are neonatal mortality, serious composite neonatal morbidity, and long-term neuro-
logic outcome.
30
There is now a randomized, placebo-controlled, double-blinded
Juul & Ferriero
122
parallel group comparison study of hypothermia and allopurinol ongoing (the European
ALBINO Trial). Allopurinol is being given twice: 30 minutes after birth and then 12 hours
later, in addition to hypothermia in moderate to severe HIE. Outcomes will be assessed
at 2 years of life.
There are many antioxidants that have been investigated in both preterm and term
HI injury. Scavengers such as melatonin and vitamin E have shown promise. Lipid
peroxidation inhibitors such as the lazaroids, gingko biloba, and caffeic acid, and
FR reducers such as ebselen and Epo, have produced some amelioration of injury.
Nitric oxide synthase inhibitors such as aminoguanidine, L-omega-nitro-arginine-
methyl-ester (L-NAME), 7 nitroindazole, and newer derivatives are still being
investigated.
31,32
The most promising of these agents seems to be melatonin, which shows efficacy in
both preterm and term injury. Melatonin has many targets along the injury cascade,
including oxidative stress, inflammation, apoptosis, mitochondrial failure, as well as
nuclear effects. The benefit is in the lack of significant side effects in children and
term neonates. A recent observational study showed that melatonin levels are deficient
in preterm and term newborn infants, and it is now being trialed daily for 7 days after
premature birth to identify whether it will reduce the risk of prematurity-associated
brain injury (MINT; ISRCTN15119574). Another investigation is underway in premature
and full-term babies to identify optimal treatment doses (MELIP, NCT01340417; and
MIND, NCT01340417), and there is a study to determine the effects of maternal sup-
plementation on outcome in term infants (PREMELIP; identification number pending).
An Australian study evaluating melatonin to prevent brain injury in unborn growth-
restricted babies is ongoing in which mothers receive melatonin during pregnancy
and oxidative stress is monitored in maternal serum, placenta, and umbilical cord
blood. A composite neonatal outcome will be evaluated (NCT01695070).
Dietary manipulations may also prove promising in neuroprotection. Pomegranate
juice is rich in polyphenols that can protect the neonatal mouse brain against an HI
insult when given to mothers in their drinking water.
33
Even when given after the insult
to neonatal animals, there is substantial protection in hippocampus, cortex, and stria-
tum.
34
Omega-3 polyunsaturated fatty acid supplementation can reduce brain dam-
age and improve long-term neurologic outcomes even 5 weeks after an HI insult to
rodents. The effect is best appreciated in microglia, where nuclear factor kappa B acti-
vation and release of inflammatory mediators are inhibited, thus providing an antiin-
flammatory effect as well.
35
Antiinflammatory Agents
As mentioned earlier, melatonin has multiple targets in the injury cascade and is a
perfect candidate for manipulating inflammation. In several animal models, small
and large, preterm and term, it has shown efficacy against excitotoxic lesions as
well as HI. A recent study in rodents revealed that melatonin preserved white matter
and learning disabilities after ibotenate lesions to the postnatal day 5 brain and was
equally efficacious against IL-1binjections.
36
In a fetal sheep model at E90, cord oc-
clusion produced substantial white matter injury that was blocked with melatonin.
37
In
term brain models of HI, melatonin markedly decreased microglial activation and pre-
served myelination.
38
The most promising study to date in piglets revealed that mela-
tonin, in combination with hypothermia, provided substantial improvement compared
with hypothermia in preserving brain function measured by amplitude-integrated elec-
troencephalogram, and reduced cell death in the thalamus, the region most affected
by the asphyxia insult.
39
There is now a collaborative study between Hopital Robert
Debre and St Thomas Hospital (Kings College London) as a proof of concept in
Strategies in Neonatal Brain Injury 123
neuroprotection study. It will be a double-blinded randomized trial of premature new-
borns of less than 28 weeks’ gestational age. Babies will be randomized to placebo,
low-dose melatonin, or high-dose melatonin, and outcome will be assessed by mag-
netic resonance imaging and neurodevelopmental outcome at 24 months (MINT Trial,
ISRCTN15119574).
GROWTH FACTORS AS NEUROPROTECTANTS
Many growth factors have essential roles during fetal and postnatal brain develop-
ment. Although the effects of some, such as brain-derived neurotrophic factor
(BDNF), are largely restricted to the brain, others such as Epo, VEGF, granulocyte col-
ony–stimulating factor (GCSF), and insulinlike growth factor 1 (IGF-1) have important
somatic effects in addition to their roles in neurodevelopment. All of the factors listed
earlier have been evaluated as neuroprotectant therapies for adult and neonatal brain
injury. At this time, Epo is the best studied for this purpose, and is the closest to clinical
use. The pleiotropic nature of these growth factors makes it essential to test meticu-
lously for safety before clinical use, particularly because very high doses are often
required for neuroprotection, given that these large molecules do not readily cross
the blood-brain barrier.
Epo
Epo and its receptor (EpoR) are expressed in the developing central nervous system
(CNS), and are required for normal brain development.
40
Acute exposure to hypoxia
upregulates the expression of EpoR on oligodendrocytes and neurons, without a
commensurate increase in Epo expression.
41
The presence of unbound cell surface
EpoR drives cells of neuronal and oligodendrocyte lineage to apoptosis, whereas
ligand-bound EpoR activates survival signaling pathways. With Epo binding, EpoR di-
merizes to activate antiapoptotic pathways via phosphorylation of JAK2, phosphory-
lation, and activation of mitogen-activated protein kinase (MAPK), extracellular related
kinase (ERK1/2), as well as the phosphatidylinositaol 3-kinase (PI3K/Akt) pathway and
signal transducer and transcriptional activator 5 (STAT5), which are critical in cell sur-
vival.
42
Epo also stimulates production of BDNF. Epo signaling inhibits early mecha-
nisms of brain injury by its antiinflammatory,
43,44
antiexcitotoxic,
45
antioxidant,
46,47
and antiapoptotic effects on neurons and oligodendrocytes. Repair of brain injury is
also enhanced in the presence of Epo because of its positive effects on neurogenesis
and angiogenesis, which are essential for plasticity and remodeling.
48,49
Epo effects
are dose dependent, with multiple doses being more effective than single doses.
50,51
Epo reduces neuronal loss and learning impairment following brain injury,
52
and, even
when initiated as late as 48 to 72 hours after injury, there is evidence of improved
behavioral outcomes, enhanced neurogenesis, increased axonal sprouting, and
reduced white matter injury in animal models of brain injury.
53,54
Epo is now under investigation for both term and preterm brain injury. The antiin-
flammatory, antiexcitotoxic, and antioxidant effects are relevant to brain injury in
both age groups. The specific effects of Epo in preoligodendrocytes may be most rele-
vant to the white matter injury that characterizes preterm brain injury. Treatment
approaches to acute brain injury in term infants (HIE) and preterm infants (intraventric-
ular hemorrhage [IVH]) should differ from preventative strategies in preterm infants. In
the former, a shorter duration of high-dose Epo is most appropriate, whereas for the
latter, a more prolonged treatment strategy that continues during the period of oligo-
dendrocyte vulnerability is most likely to succeed. In addition to the specific cellular
effects on neurons and oligodendrocytes, this more prolonged treatment also
Juul & Ferriero
124
decreases the availability and potential toxicity of free iron, caused by the erythropoi-
etic effects of Epo, by increasing iron utilization.
Translation to Clinical Trials
Epo does not cross the placenta, so prenatal treatment is not an option. It is approved
by the US Food and Drug Administration and has a robust safety profile in neonates,
with more than 3000 neonates randomized to placebo-controlled trials testing its eryth-
ropoietic effects.
55
Doses required for neuroprotection are higher than those used for
prevention and treatment of anemia, because only a small fraction of circulating Epo
crosses the blood brain barrier. In animal models of neonatal brain injury, Epo doses
of 1000 to 5000 U/kg result in sustained neuroprotection, improving both short-term
and long-term structure and function.
56
Phase I/II trials have been done to establish
safety and translational pharmacokinetics of Epo in preterm
57,58
and term neonates.
59
These studies suggest that 1000 U/kg/dose provides an area under the curve (AUC)
most similar to a neuroprotective dose of 5000 U/kg in rodents (Table 2). The optimal
dose and duration of treatment is likely to differ for treatment of HIE compared with pre-
venting or treating brain injury in preterm infants, and is not yet known. Note that the
pharmacokinetics in preterm and term asphyxiated infants are different, with a longer
half-life noted at higher doses in infants with HIE. Phase II and III studies are now un-
derway for neuroprotection of both extreme prematurity and HIE in term infants
(Box 1). In the United States, a multicenter randomized controlled trial of preterm
Epo neuroprotection is beginning (PENUT trial, NCT01378273). This study will use
1000 U/kg for 6 doses followed by 400 U/kg 3 times a week until 33 weeks of gestation.
A Swiss trial has used 3000 U/kg for 3 doses in the first weeks of life. Enrollment is com-
plete for this trial, with follow-up underway. The BRITE study, comparing Darbepoetin,
Epo, and placebo, is showing improved outcomes in preterm neonates receiving either
Darbepoetin or Epo. Erythropoietic agents (Epo and darbepoetin) are also being stud-
ied in combination with hypothermia for the treatment of HIE in term infants. Pilot
studies have shown safety and early signs of benefit, and larger studies are planned
or ongoing in the United States, France (NCT01732146), and China.
BDNF
BDNF is an important growth factor during fetal brain formation, particularly in the hip-
pocampus, cerebral cortex, basal forebrain, and cerebellum. It is also active in adult
neurogenesis. BDNF binds primarily to receptor tyrosine kinase B and activates
MAPK and Ca
21
-calmodulin kinase II (CAMKII), which regulate cAMP-responsive
Table 2
Epo pharmacokinetics
Epo Dose (U/kg) AUC C
max
T
1/2
P7 Rodents SC 5000 117,677 6224 8.4
P7 Rodents IP 5000 140,331 10,015 6.7
Preterm infants <1000 g 500 31,412 2780 8078 538 5.4 0.6
1000 81,498 7067 14,017 1293 7.1 0.7
2500 317,881 22,941 46,467 2987 8.7 1.4
Term HIE infants 500 50,306 67,426 7046 814 7.2 1.9
1000 131,054 17,083 13,780 2674 15.0 4.5
2500 328,002 61,945 33,316 7377 18.7 4.7
Abbreviations: AUC, area under the curve; C
max
, maximum plasma concentration of the drug; IP,
intraperitoneal; P7, postnatal day 7; SC, subcutaneously; T
1/2
, half life.
Strategies in Neonatal Brain Injury 125
element binding (CREB) and synapsin transcription. Neuroprotection from glutamate
toxicity is mediated through PI3K and the Ras/MAPK signaling pathways, and involves
an increase in B-cell lymphoma 2 (bcl-2) proteins.
60
Both exercise and caffeine in-
crease BDNF secretion, thereby increasing recognition memory and neurogenesis.
VEGF
VEGF is a growth factor that is stimulated by HIF-1 and stimulates vasculogenesis and
angiogenesis, essential processes needed for brain development and brain repair. In
addition, VEGF has specific neurotrophic and neuroprotective effects in adult and
neonatal models of hypoxic-ischemic brain injury.
61
These effects also involve activa-
tion of Akt and extracellular receptor kinase (ERKs). Tight regulation of this factor is
needed because both overexpression and underexpression can contribute to disease.
GCSF
GCSF is a hematopoietic glycoprotein that stimulates the clonal maturation of neutro-
phil progenitors and increases many functional activities of mature neutrophils. In
addition to these hematopoietic effects, GCSF and its receptor are expressed on
neuronal cells in a variety of brain regions.
62
GCSF has shown neuroprotection in
several models of brain injury, and is well tolerated at high doses. GCSF has been
Box 1
Clinical trials of neuroprotective agents
Phase 1 and 2 trials
Xenon and cooling therapy in babies at high risk of brain injury following poor condition at
birth: randomized pilot study. Bristol, United Kingdom
Phase 2 and 3 trials
Safety and efficacy of topiramate in neonates with HIE treated with hypothermia (NeoNATI),
Florence, Italy. NCT01241019
Neonatal Epo and therapeutic hypothermia; short-term outcome study (NEAT O Study).
University of California, San Francisco, multicenter, Thrasher funded
Darbepoetin administration in newborns undergoing cooling for encephalopathy (DANCE
Study) Utah, multicenter, Thrasher funded. NCT01471015
Phase 3 trials
A multicenter randomized controlled trial of therapeutic hypothermia plus magnesium
sulfate (MgSO
4
) versus therapeutic hypothermia plus placebo in the management of term
and near-term babies with HIE. Turkey. NCT01646619
Optimizing cooling strategies at less than 6 hours of age for neonatal HIE. A National
Institute of Child Health and Human Development (NICHD)–funded project. NCT01192776
Phase III study of efficacy of high-dose Epo to prevent HIE sequelae in term newborns. Paris,
France. NCT01732146
Evaluation of systemic hypothermia initiated after 6 hours of age in infants of greater than
or equal to 36 weeks’ gestation with HIE: a bayesian evaluation. An NICHD-funded project.
NCT00614744
Preterm Epo Neuroprotection (PENUT) Trial. A multicenter, randomized, placebo-controlled
phase III 940–subject trial of Epo for the neuroprotection of extremely low gestational age
neonates. An NINDS-funded project. NCT01378273
Melatonin as a Novel Neuroprotectant in Preterm Infants Trial (MINT). UK trial funded by
Medical Research Council. ISRCTN15119574
Juul & Ferriero
126
reported to have antiapoptotic, antiinflammatory, antiexcitotoxic, and neurotrophic
properties with demonstrated improved long-term outcomes.
63
It is currently being
evaluated in a phase II clinical trial for adult ischemic stroke (NCT00132470).
64
IGF-1
IGF-1 has an important role in normal brain development, promoting neuronal growth,
cellular proliferation, and differentiation in vitro, when injected directly into brain, or
when given intranasally.
65
It has also been found to have neuroprotective effects,
improving long-term function after hypoxic-ischemic brain injury. However, its clinical
application to neurologic disorders is limited by its large molecular size, poor central
uptake, and mitogenic potential.
Drug Delivery
When considering drugs for neuroprotection, drug delivery is an important consider-
ation. For example, if a drug crosses the placenta and can be tolerated by the mother,
prenatal treatment is an option, such as with xenon and melatonin. The ability to give
the drug intravenously is important, given that many critically ill neonates cannot take
oral medications. Some medications, such as melatonin, are presently only available
as oral formulations. Inhalational agents such as xenon have the problem that, if a sig-
nificant oxygen requirement exists, the ability to deliver neuroprotective concentra-
tions (30%–50%) may be limited.
Box 2
Gaps in knowledge
1. Cytokine response
Innate immunity differs in newborns compared with adults
Th1 preponderance in newborns
Th2 in adults
When does it change?
How is this affected by prematurity?
How does this affect brain injury/repair?
2. Microglial response
Three activation states of CNS microglia
M1: classic activation (tissue defense, proinflammatory)
M2: alternative activation (repair, antiinflammatory, fibrosis, matrix reconstruction)
M3: acquired deactivation (immunosuppression, phagocytosis of apoptotic cells)
How are these states regulated?
Can this response be harnessed for healing?
Does it differ for preterm versus term infants?
3. Preconditioning
Hypoxia
Inflammation
What are the molecular mechanisms?
Can these mechanisms be harnessed to improve outcomes?
Strategies in Neonatal Brain Injury 127
Small neuroactive peptides may have a therapeutic advantage compared with
larger molecules such as Epo or GCSF, which are larger glycoproteins that do not
cross the blood-brain barrier well. Modified neuropeptides and mimetic peptides
have been designed to overcome these barriers, and are in various stages of testing.
Another novel approach is to use alternative delivery methods, targeting the inflam-
matory system. Polyamidoamine dendrimers have been shown to localize in activated
microglia and astrocytes in the brains of newborn rabbits with CP, but not healthy con-
trols.
66
This nanotechnology approach has been used with excellent results to deliver
dendrimer-bound N-acetyl-L-cysteine (NAC) to the target inflammatory cells to sup-
press neuroinflammation, using much lower concentrations than are needed with sys-
temic dosing.
Combination Therapies
As more information is gained about mechanisms of brain injury and neuroprotection,
combination therapies may be applied. As mentioned earlier, some preclinical combi-
nation studies are already underway, for example, hypothermia plus xenon, and hypo-
thermia plus Epo for the treatment of term HIE.
Gaps in Knowledge
There remain gaps in knowledge (Box 2). All of these must be considered in the
context of the developing neonate, because immune function, cell populations, and
specific vulnerabilities and response to injury change over time. As clinicians become
more knowledgeable in these areas, new approaches to neuroprotection may become
apparent.
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