Caffeine for apnea of prematurity: Effects on the developing

brain
Author links open overlay panelAnzariAtikaRichardHardingaRobertDe MatteoaDelphiKondos-
DevcicbJeanieCheongcLex W.DoylecMaryTolcosbde
Show more

https://doi.org/10.1016/j.neuro.2016.11.012Get rights and content

Highlights
•
Caffeine is widely used to treat apnea of prematurity (AOP) in preterm
infants.

•
Caffeine is an adenosine receptor antagonist.

•
Caffeine can have beneficial or adverse effects on the developing brain.

•
However, most studies suggest that caffeine is detrimental to the
developing brain.

•
The maximal dose of caffeine that is safe to use for AOP needs to be
determined.

Abstract
Caffeine is a methylxanthine that is widely used to treat apnea of prematurity
(AOP). In preterm infants, caffeine reduces the duration of respiratory support,
improves survival rates and lowers the incidence of cerebral palsy and
cognitive delay. There is, however, little evidence relating to the immediate
and long-term effects of caffeine on brain development, especially at the
cellular and molecular levels. Experimental data are conflicting, with studies
showing that caffeine can have either adverse or benefical effects in the
developing brain. The aim of this article is to review current understanding of
how caffeine ameliorates AOP, the cellular and molecular mechanisms by
 which caffeine exerts its effects and the effects of caffeine on brain
development. A better knowledge of the effects of caffeine on the developing
brain at the cellular and/or molecular level is essential in order to understand
the basis for the impact of caffeine on postnatal outcome. The studies
reviewed here suggest that while caffeine has respiratory benefits for preterm
infants, it may have adverse molecular and cellular effects on the developing
brain; indeed a majority of experimental studies suggest that regardless of
dose or duration of administration, caffeine leads to detrimental changes
within the developing brain. Thus there is an urgent need to assess the impact
of caffeine, at a range of doses, on the structure and function of the
developing brain in preclinical studies, particularly using clinically relevant
animal models. Future studies should focus on determining the maximal dose
of caffeine that is safe for the preterm brain.
 Previous article in issue
 Next article in issue

Keywords
Prematurity
Apnea
Caffeine
Adenosine
Brain
Development

1. Apnea of prematurity
Owing to immaturity of the lungs and other organs, preterm infants are
susceptible to respiratory and metabolic abnormalities, which can result in
hypoxemia, acidemia and hypoglycaemia. In spite of advances in neonatal
intensive care, preterm infants are at risk of respiratory and neurological
sequelae, with the risk being highest in those born very preterm (<32 weeks’
gestational age) or extremely preterm (<28 weeks’ gestational age) (Doyle et
al., 2010b; Fanaroff et al., 2007). A common problem experienced by very and
extremely preterm infants is apnea of prematurity (AOP), which is defined as a
cessation in breathing lasting more than 15–20 s (Barrington and Finer, 1991;
Miller and Martin, 2011). AOP occurs in 85% of infants born prior to 34 weeks’
gestational age (Barrington and Finer, 1991). The incidence of AOP is
inversely correlated with gestational age, occurring in 7% of infants born at
34–35 weeks, 15% at 32–34 weeks, 54% at 30–31 weeks, and in nearly all
infants born prior to 30 weeks or with a birth weight <1000 g (Henderson-
Smart, 1981; Robertson et al., 2009). If untreated, AOP can lead to
hypoxemia, which can result in tissue hypoxia and hypoxic organ injury.
Cessation of respiratory airflow (apnea) can be caused by a cessation of the
central respiratory rhythm, airway obstruction, or a combination of these.
Thus, three main types of apnea are recognised: i) central apnea,
characterised by a cessation of inspiratory efforts in the absence of airway
obstruction, ii) obstructive apnea, when the infant attempts to breath against
an obstructed upper airway, and iii) mixed apnea, when inspiratory efforts are
obstructed, usually following periods of central apnea (Martin et al., 2004;
Milner et al., 1980). AOP is believed to be a result of immaturity of respiratory
control mechanisms (e.g. reduced sensitivity to CO2 and hypoxia) as well as
an exaggerated protective (laryngeal closure) response to laryngeal
stimulation; recent evidence suggests that inflammation in the central nervous
system (CNS) may also play a role (Morton and Smith, 2016).
The central respiratory rhythm is dependent upon input from chemoreceptors
near the ventral surface of the medulla oblongata that respond to the ambient
pH and partial pressure of carbon dioxide (PaCO2); the rhythm is also affected
by excitatory and inhibitory inputs from higher brain centres,
mechanoreceptors in the upper airway and lungs, and chemoreceptors in the
carotid bodies (Di Fiore et al., 2013; Martin and Wilson, 2012; Mathew, 2011).
A cessation of the respiratory rhythm in the brainstem (central apnea) plays
an important causative role in AOP as it will lead to a cessation in the
inspiratory activation of the muscles of respiration, including the diaphragm,
intercostal muscles and dilator muscles of the upper airway (larynx, pharynx,
and tongue). As well as a cessation of inspiratory efforts, the upper airway
may become closed; indeed it is likely that during central apnea, the glottis
becomes actively closed, as it is in the fetus during periods of central apnea
(Harding, 1984). In preterm infants, the soft tissues of the respiratory tract are
highly compliant, predisposing these infants to upper airway collapse and
obstruction during inspiratory efforts, especially in the absence of dilator
muscle activity (Di Fiore et al., 2013).
2. Caffeine for the treatment of apnea of prematurity
Since the 1970s, methylxanthines, including caffeine, theophylline and
aminophylline (the ethylenediamine salt of theophylline) have been the
pharmacological drugs of choice for the treatment of AOP. Methylxanthines,
combined with the prone sleeping position, continuous positive airway
pressure or nasal intermittent positive pressure ventilation, comprise the
current standard of care for AOP. Methylxanthines are able to reduce the
incidence of apneic episodes via a number of pathways and have therefore
become one of the most commonly prescribed drugs in neonatal medicine
(Millar and Schmidt, 2004). Methylxanthines are believed to act by raising
central sensitivity to CO2 and improving respiratory muscle function, which
together lead to an increase in minute ventilation (Abu-Shaweesh and Martin,
2008; Miller and Martin, 2011). Although it is well established that
methylxanthines lead to an increase in respiratory neural output, the
molecular and cellular basis of this effect is still unclear. The hydrophobic
properties of caffeine allow it to pass through all biological membranes,
including the blood-brain barrier (Lachance et al., 1983); thus it readily enters
the CNS. The ability of methylxanthines to competitively antagonise
adenosine receptors (ARs) within the CNS, in particular A1 and A2A ARs, has
been proposed as the mechanism by which these agents stimulate the
respiratory rhythm (Fredholm, 1995). A secondary mechanism may be via the
effects of methylxanthines on gamma-aminobutyric acid (GABA) receptors,
inhibition of phosphodiesterase (PDE) and calcium (Ca2+) release; however
these latter effects are unlikely to be seen owing to the extremely high (toxic)
concentrations that are required (Fisone et al., 2004; Fredholm et al., 1999).
Numerous studies have compared the benefits and risks of methylxanthines in
preterm infants. Although aminophylline and theophylline are just as effective
in treating AOP as caffeine, they are associated with far more adverse effects
than caffeine (Henderson-Smart and Steer, 2010; Larsen et al., 1995). Some
of the well documented side-effects of methylxanthines include tachycardia,
cardiac dysrhythmias, food intolerance, increased metabolic rate, increased
O2 consumption, and less frequently, seizures, all of which are uncommon
with current therapeutic doses of caffeine (Abu-Shaweesh and Martin, 2008).
Another advantage of caffeine is that it is more easily absorbed than
aminophylline and theophylline, and has a wider therapeutic range and a
longer half-life, allowing for once-a-day dosing (Henderson-Smart and De
Paoli, 2010; Henderson-Smart and Steer, 2010; Millar and Schmidt, 2004).
These benefits, along with the finding that caffeine improves respiratory and
neurodevelopmental outcomes up to 21 months of age (Schmidt et al., 2006,
2007), have led to caffeine being the methylxanthine of choice when treating
AOP. However, the dosing regimen of caffeine used in treating preterm infants
varies between neonatal intensive care units around the world (Scanlon et al.,
1992; Steer et al., 2003), and higher doses of caffeine are often administered
when the standard dose is not sufficient to reduce the incidence of apnea. In
addition, the timing of caffeine therapy in relation to birth is an important
consideration (Schmidt et al., 2014). A preliminary randomized controlled trial
of 21 neonates born at less than 29 weeks’ gestational age who received
caffeine (20 mg/kg) either prior to 2 h after birth or at 12 h after birth found no
difference in ventilatory requirements; however, their hemodynamics improved
more after early caffeine treatment compared with later treatment (Katheria et
al., 2015). Whether early versus late caffeine therapy in preterm infants
differentially affects brain development or the incidence of neonatal brain
injury is yet to be determined.
3. Caffeine: mechanism of action
As caffeine has a range of molecular targets within the CNS, it has been
difficult to determine the precise molecular and cellular mechanisms by which
caffeine reduces the incidence of AOP. The major molecular target of caffeine
within the CNS is antagonism of ARs, particularly the A1 and A2A receptors; at
high concentrations, caffeine leads to the inhibition of PDE, release of
intracellular Ca2+ and antagonism of GABAA receptors. The effect of clinical
levels of caffeine on arousal and breathing is unlikely to be a result of its
actions on PDE, intracellular Ca2+ and GABAA receptors (Fredholm et al.,
1999), as these targets require very high concentrations of caffeine (in the
millimolar (mM) range) to achieve an effect greater than 80% of the maximal
effect (Fig. 1) (Fisone et al., 2004; Fredholm et al., 1999); such high
concentrations are rarely reached in humans. Indeed a blood caffeine
concentration of 500 μM is considered sufficient to cause lethal intoxication.
Importantly, administration of the standard dose of caffeine (loading dose:
20 mg/kg; maintenance dose: 5–10 mg/kg) leads to a peak plasma caffeine
concentration of 84 to 128 μM in the neonate (Aranda et al., 1979; Charles et
al., 2008), supporting the notion that the respiratory stimulatory action of
caffeine and its arousal effects on the CNS are due to antagonism of ARs
rather than the other molecular targets (Abu-Shaweesh, 2007; Fredholm,
1995; Herlenius and Lagercrantz, 1999); therefore the role of caffeine in
inhibiting PDE, releasing intracellular Ca2+ and antagonising GABAA receptors
will not be discussed further in this review.

1. Download high-res image (241KB)

2. Download full-size image
Fig. 1. Concentration of circulating caffeine required to activate molecular
targets in relation to human caffeine consumption.
One cup of coffee, leading to a circulating caffeine concentration of ∼10 μM, is
sufficient to have a significant effect on A1 and A2A adenosine receptors.
However, 20-times higher concentrations of caffeine are required to inhibit
PDE; 40-times higher concentrations are needed to block GABAA receptors,
and 100-times higher concentrations are required to lead to Ca2+ release.
Millimolar (potentially toxic) concentrations of caffeine are required to
significantly affect these later mentioned molecular targets. Figure adapted
from (Bonati et al., 1982; Dews, 1982; Fredholm et al., 1999).
3.1. Role of adenosine and its receptors
Adenosine is a normal constituent of cells and numerous enzymes regulate its
intracellular concentration. Adenosine serves many diverse roles in normal
physiology including the control of neuronal excitability, which affects the state
of central arousal and the promotion or maintenance of sleep (Portas et al.,
1997; Rainnie et al., 1994); adenosine is also involved in the coupling of
cerebral blood flow to energy demand (Dirnagl et al., 1994; Ko et al., 1990).
Intracellularly, adenosine can be formed via the hydrolysis of S-
adenosylhomocysteine (SAH); however, it is predominantly formed from the
degradation of adenosine monophosphate (AMP) by AMP selective 5′-
nucleotidase, with the rate of adenosine formation related to the amount of
available AMP (Fig. 2) (Fredholm et al., 1999; Latini and Pedata, 2001). The
subsequent metabolism of adenosine occurs within the cell, following the
uptake of extracellular adenosine into the cell; within the cell, adenosine is
metabolised by phosphorylation to AMP via adenosine kinase (AK) or via
deamination to inosine by adenosine deaminase (ADA) (Latini and Pedata,
2001).

1. Download high-res image (247KB)

2. Download full-size image
Fig. 2. Adenosine formation, transport and metabolism.
Pathways of adenosine formation, transport and metabolism are indicated by
arrows. Intracellularly, 5′-AMP, formed via cyclic AMP, is degraded by 5′-
nucleotidase to form adenosine. Intracellular adenosine synthesis can also
occur via the hydrolysis of SAH. Similarly, extracellular adenosine synthesis
occurs via the degradation of 5′-AMP by Ecto-5′-nucleotidase. The
subsequent metabolism of adenosine takes place within cells following the
transport of extracellular adenosine into the cell; here, adenosine is
phosphorylated to 5′-AMP via AK or deaminated to inosine by ADA. ADA,
adenosine deaminase; ADP, adenosine diphosphate; AK, adenosine kinase;
AMP, adenosine monophosphate; ATP, adenosine triphosphate; PDE,
phosphodiesterase, SAH, S-adenosylhomocysteine. Figure adapted from
(Latini and Pedata, 2001).
Four distinct ARs have been cloned and characterised; these receptors are
designated A1, A2A, A2B and A3. A3 and A2B ARs require much higher
concentrations of adenosine for activation, whereas A1 and A2A ARs are
activated at low concentrations of adenosine (Fredholm et al., 1999) and are
therefore likely to be the major targets for caffeine. Caffeine has a similar
molecular structure to adenosine (Fig. 3) and thus the majority of the research
on caffeine’s effects on adenosine receptors has focussed on A1 and A2A ARs.

1. Download full-size image

Fig. 3. Molecular structures of caffeine and adenosine.
A comparison of the molecular structures of caffeine and adenosine shows
that both compounds share a similar double ring structure. Figure adapted
from (Fredholm, 2011).
A1 and A2A ARs are G-protein-coupled receptors with opposing actions at the
cellular level. A1AR activation leads to the inhibition of adenylyl cyclase and
some types of voltage sensitive Ca2+ channels (e.g. N- and Q- channels) and
activation of several types of potassium channels such as phospholipase C
and D, which leads to numerous cellular effects (Fredholm et al., 1999). On
the other hand, A2AAR activation leads to the activation of adenylyl cyclase
and some types of voltage sensitive Ca2+ channels, especially L-channels
(Fredholm et al., 1999). Thus A1 and A2A ARs are considered to have partly
opposing actions at the cellular level (Fredholm et al., 1999). A1ARs are quite
widespread and present in almost all brain areas, with highest abundance in
the hippocampus, cerebral and cerebellar cortex and certain thalamic nuclei
(Fastbom et al., 1987; Goodman and Synder, 1982), and moderate
expression in the striatum (Fredholm et al., 1999). There is evidence to
suggest that A1ARs are located on nerve terminals rather than neural cell
bodies (Johansson et al., 1993); however, other studies have shown these
receptors to be located on neuronal cell bodies, oligodendrocytes, microglia,
astrocytes and axons, with abundant mRNA expression of these ARs in white
matter tracts (Cunha, 2005; Reppert et al., 1991; Swanson et al., 1995).
A2AARs are expressed in only a few regions of the brain; they are mainly
concentrated in the dopamine-rich regions, including the striatum, nucleus
accumbens, with very little mRNA detected in the hippocampus, cortex and
medulla oblongata (Dunwiddie and Masino, 2001; Fredholm et al., 1999).
A2AARs appear to be located on cell bodies of GABAergic output neurons and
are also co-localised with dopamine D2 receptors on medium-sized spiny
neurons in the dorsal striatum (Fink et al., 1992; Johansson et al., 1993;
Schiffmann et al., 1991), as well as with microglia and astrocytes (Cunha,
2005).
In the past, the stimulation of breathing by caffeine and its ability to cause
CNS arousal were attributed to effects of caffeine on the A1AR, as the protein
for this receptor is expressed in the brainstem; furthermore, adenosine-
induced activation of A1AR exerts inhibitory effects on inspiratory neurons
(Herlenius et al., 2002Herlenius and Lagercrantz, 1999). This inhibitory effect
is induced via pathways that include inhibition of excitatory glutamatergic
synaptic transmission, decreased Ca2+ current, inhibition of intracellular cyclic
adenosine monophosphate (cAMP) production and facilitation of potassium
conductance (Zaidi et al., 2006). However, studies now suggest that A2AARs
may also be involved and that the central arousal effects of caffeine may be
mediated by a combination of its effects on both the A1and A2A receptors
(Dunwiddie and Worth, 1982; Satoh et al., 1999). By using gene deletion
strategies in rats to silence the expression of A2AAR, it has been shown that
A2AARs in the shell region of the nucleus accumbens are responsible for the
CNS arousal effects of caffeine (Lazarus et al., 2011). Furthermore, a
subpopulation of GABAergic neurons that express A2AAR mRNA has been
located in the medulla oblongata (Wilson et al., 2004; Zaidi et al., 2006) and it
is known that GABAergic pathways contribute greatly to the inhibition of
inspiration that characterises some respiratory reflexes in early postnatal life
(Abu-Shaweesh et al., 2001; Mayer et al., 2006). Thus, adenosine and GABA
may operate synergistically to modulate respiratory neural output.
In the developing brain, A1ARs are expressed during periods of neurogenesis,
neuronal migration and axonal sprouting (Rivkees, 1995), and increased
receptor activation during these periods can cause cell death (Turner et al.,
2002a). Indeed, neonatal rats treated with an A1AR agonist (N6–
cyclopentyladenosine) show marked reductions in white and grey matter
volume with secondary ventriculomegaly, along with reduced total axonal
volume and reduced myelination (Turner et al., 2002b). As oligodendrocytes
express A1ARs, the adverse effects of A1AR agonists or sustained activation
of the A1 receptor on myelination is most likely a consequence of alterations to
oligodendrocytes (Rivkees and Wendler, 2011). For example, sustained
activation of A1ARs inhibits oligodendrocyte progenitor cell proliferation,
resulting in fewer oligodendrocytes, and subsequently hypomyelination
(Rivkees and Wendler, 2011; Stevens et al., 2002). On the other hand,
antagonism of A1ARs could offer a neuroprotective effect (Rivkees and
Wendler, 2011), as caffeine treatment of neonatal mice reared in a hypoxic
environment was associated with reduced ventriculomegaly and
hypomyelination along with an increased proportion of immature
oligodendrocytes (Back et al., 2006).
Activation of A2AARs contributes to ischaemic tissue damage (Chen et al.,
2007; Cunha, 2005). Stimulation of A2AARs by adenosine causes activated
microglia to assume their characteristic amoeboid morphology during brain
inflammation (Orr et al., 2009); caffeine, an A2AAR antagonist, prevents this
effect and subsequently prevents neuroinflammation (Rebola et al., 2011).
However, the mechanisms by which A2AAR antagonism protects the
developing brain against damage are largely still unknown (Rebola et al.,
2011). This protection may be a result of A2AARs playing a key role in
controlling microglial activation and the secretion of brain-derived neurotrophic
factor (BDNF) upon microglial activation (Gomes et al., 2013). While it
appears that the neuroprotective effects of caffeine are mediated either via the
A1 or A2AARs, it should be noted that, to date, studies using transgenic mice
have not revealed that a single class of AR mediates this neuroprotective
effect, as the protective effects of caffeine are seen in A1, A2A, A2B and
A3 knockout mice (Rivkees and Wendler, 2011).
3.2. Effects of caffeine on the developing central nervous system

3.2.1. Clinical studies

Several clinical trials have assessed the efficacy of caffeine in treating AOP
and the effects of caffeine on neurodevelopmental outcomes. The “Caffeine
for AOP” (CAP) trial was one of the largest international randomized
controlled trials in preterm neonates. This study showed that preterm infants
treated with caffeine citrate (20 mg/kg loading dose; 5–10 mg/kg maintenance
dose) had improved rates of survival without neurodevelopmental disability,
and lower rates of cerebral palsy and cognitive delay at 18–21 months of age
compared with placebo-treated preterm infants (Schmidt et al., 2006, 2007).
When these infants were assessed at 5 years of age, neonatal caffeine
therapy was associated with improved motor function but was no longer
associated with significantly improved rates of cerebral palsy or intellectual
impairment (Schmidt et al., 2012). In a subgroup of infants in the CAP trial
who underwent magnetic resonance imaging at term corrected age, caffeine
treatment improved cerebral white matter development as evidenced by more
mature organization of cerebral white matter (Doyle et al., 2010a).
Subsequent to the CAP trial, other studies have assessed whether higher
doses of caffeine confer additional beneficial effects to both the preterm brain
and neonatal neurobehavioural outcomes. Of concern, a recent pilot
randomized trial in preterm infants born at ≤30 weeks’ gestational age treated
with either a highdose (80 mg/kg) or a standard dose (20 mg/kg) of caffeine
citrate within the first day after birth, reported a significantly increased
incidence of cerebellar hemorrhage, more hypertonicity and greater deviant
neurologic signs at term equivalent age following the higher dose (McPherson
et al., 2015). However, in another cohort of preterm infants, high-dose
neonatal caffeine citrate therapy (80 mg/kg loading dose; 20 mg/kg
maintenance dose) was not associated with adverse neurological outcomes
including development, temperament and behaviour, at 1 and 2 years of age
(Gray et al., 2011). Thus, there is a need for further research on the effects of
higher doses of caffeine on the preterm brain to determine their safety from a
neurobehavioural perspective.
The effects of caffeine administration on cerebral perfusion in preterm infants
have been assessed in some studies. Specifically, acute low-dose caffeine
citrate treatment (2.5 mg/kg/day) in preterm infants (<32 weeks’ gestational
age) had no effect on cerebral oxygenation, measured by near-infrared
spectroscopy on the third day of caffeine administration, with measurements
taken 30 min before to 60 min after caffeine treatment (Dani et al., 2000). In
another study, caffeine citrate administered (10 mg/kg) to preterm infants (<34
weeks’ gestational age) reduced cerebral oxygenation and cerebral blood flow
velocity 1 h after caffeine treatment, with a partial recovery at 4 h (Tracy et al.,
2010). Functionally, a loading dose of caffeine citrate (10 mg/kg; i.v) increased
the amplitude-integrated electroencephalogram in preterm infants (<34 weeks’
gestational age) during the first 2 h following administration; the authors
consider that these changes are an indication of enhanced cortical cerebral
activity and altered arousal pattern (Supcun et al., 2010). It is likely that
differences in outcomes following caffeine treatment may be due to
differences in dosing, the age of infants at the time of assessment, as well as
the time of assessment in relation to the administration of caffeine.
An observational prospective study using data collected from 26 preterm
infants (≤30 weeks gestational age) has reported that serum caffeine levels
outside of the therapeutic range of 10–20 μg/mL, correlate with a pro-
inflammatory cytokine profile including increased plasma levels of interleukin
(IL)-1β, IL-6 and tumour necrosis factor (TNF)-α, as well a decrease in the
level of the anti-inflammatory cytokine IL-10 (Valdez et al., 2011). This is
particularly concerning, given that systemic inflammation is known to
adversely impact on the developing brain, and such a pro-inflammatory
cytokine profile in preterm infants treated with higher doses of caffeine may
account for the detrimental effects in the cerebellum (McPherson et al., 2015).
Indeed, subgroup analysis using 11 of the 26 preterm infants showed that in
infants who later developed bronchopulmonary dysplasia (BPD), plasma IL-1β
and IL-6 concentrations were higher, as was the ratio of these cytokines to IL-
10 concentrations (Valdez et al., 2011). In addition the median caffeine levels
at 1 week were not different between infants who did, or did not, develop
BPD; however, the higher caffeine levels were predominantly found in infants
who developed BPD. Although tenuous, these data suggest a possible link
between high caffeine plasma levels, an imbalance between the pro- and anti-
inflammatory prolife, and the later development of BPD in preterm infants.
Whether a similar link exists for adverse neurological outcomes is not known.
The finding that higher doses of caffeine are associated with a pro-
inflammatory profile in preterm infants also raises an important question – is
there an interaction between high dose caffeine and co-morbidities of preterm
birth including patent ductus arteriosus, respiratory distress syndrome, or
oxidative stress that can result from the requirement for additional ventilation?
These issues have not yet been resolved in clinical or preclinical studies.
3.2.2. Experimental studies
A number of experimental studies have investigated the impact of caffeine on
the developing brain using both altricial and precocial species (Table 1).
These studies show that caffeine has either a beneficial effect, including a
neuroprotective effect following neonatal hypoxia-ischaemia, a detrimental
effect, or no effect; however, interpretation of the findings is made difficult by
the differing treatment regimens including differences in dose, duration, age at
exposure, species as well as whether caffeine base or caffeine citrate was
used; as caffeine citrate contains anhydrous citric acid and 50% anhydrous
caffeine base, the dose of caffeine base is approximately half that of caffeine
citrate. It is therefore important that dosing regimens that more closely align
with those used in clinical practice be studied in species in which the timing of
major stages of brain development are more similar to the human (e.g. sheep,
non-human primate). In addition, as discussed above, caffeine is administered
to preterm infants with AOP and other co-morbidities that can often lead to
systemic inflammation and oxidative stress; however, there are no animal
studies that have examined the effects of this interaction on the developing
brain.
Table 1. Effects of caffeine on brain development: animal models.

Route of administration Caffeine dose Effects on postnatal brain References

Beneficial effects
Caffeine citrate
Decreased seizure susceptibility to Guillet and
administration (gavage) to Moderate (15–20 mg/kg)
some chemo-convulsants. Dunham (1995)
rat pups from P2-P6
Increased total dendritic length and
Caffeine citrate
arborization of layer III pyramidal Juarez-Mendez et
administered (s.c.) to rat High (50 mg/kg)
neurons of the prefrontal cortex at P35 al. (2006)
pups from P1-P12
and P70.

Detrimental effects
Increased RNA and protein content of
Caffeine citrate Low (20%
cerebellum. Yazdani et al.
supplemented protein diet protein + 1 mg/100 g body
Decreased total brain weight and (1988)
fed to rats from P0-P43 weight caffeine)
overall DNA and RNA content.
Route of administration
Caffeine citrate
administered to newborn
rats (via lactating dams)
from P0-P10
Caffeine base administered
(i.p) to mice pups from P3-
P10
Caffeine citrate
administered (gavage) to
newborn rats from P2-P6
Caffeine citrate
administered (gavage) to
newborn rats from P2-P6
Caffeine citrate
administered (gavage) to
newborn rats from P2-P6
Caffeine citrate
administered (gastric
intubation) to rat pups (P2-
P17 or P2-P20)
Caffeine citrate
administered (i.p) to rat
pups at P7
Caffeine administered (s.c)
to rat pups at P3
No adverse effects
Caffeine base administered
to fetal sheep (via maternal
circulation) from 0.7 to 0.8
of term
Neuroprotective effects
Caffeine citrate
administered (via lactating
dams) to hypoxic-ischemic
rat pups from P0-P12
Caffeine citrate
administered (via lactating
Caffeine dose Effects on postnatal brain References
Decreased cerebellar weight.
Low (4 mg/100 g body Yazdani et al.
Increased saturated fatty acid
weight) (2004)
concentration of cerebellum.
Decreased cell proliferation in the
Moderate (10 mg/kg subventricular zone and dentate gyrus
Desfrere et al.
loading dose, 2.5 mg/kg at P7.
(2007)
maintenance dose) Decreased astrocytogenesis in cerebral
cortex and white matter at P15.
Moderate (20 mg/kg
Increased A1AR binding in Guillet and
loading dose, 15 mg/kg
cerebellum, cortex and hippocampus. Kellogg (1991)
maintenance dose)
Moderate (20 mg/kg
Decreased A1AR binding in the Etzel and Guillet
loading dose, 15 mg/kg
molecular layer of cerebellum. (1994)
maintenance dose)
Increased A1AR labelling in anterior
hypothalamic area, ventromedial
Moderate (20 mg/kg hypothalamic nucleus, parabrachial
Gaytan and Pasaro
loading dose, 15 mg/kg complex and ventrolateral medulla.
(2012)
maintenance dose) Increased A2AAR labelling in ponto-
medullary nuclei and hypothalamic
areas.
Fuller et al.
High (40 mg/kg and
Delayed cerebral myelin synthesis. (1982); Fuller and
80 mg/kg)
Wiggins (1981)
Increased apoptosis in cerebral
High (3 doses at 50 mg/kg) hemispheres via caspase-3 -dependent Kang et al. (2002)
mechanisms.
Increased apoptosis in various brain
High (100 mg/kg) regions including cerebral cortex and Black et al. (2008)
caudate nucleus.
High (25 mg/kg loading No effect on developing white or grey
dose; 20 mg/kg matter, including glial cell density, Atik et al. (2014)
maintenance dose) myelination or neuronal cell density
Enhanced myelination and reduced
Low (300 mg/L) Back et al. (2006)
ventriculomegaly.
Decreased neuronal necrosis and
Low (300 mg/L) Bona et al. (1995)
infarction.
Route of administration Caffeine dose Effects on postnatal brain References
dams) to hypoxic-ischemic
rat pups from P1-P7

Caffeine citrate contains anhydrous citric acid and 50% anhydrous caffeine base; thus the dose of
caffeine base is approximately half that of caffeine citrate.

AR, adenosine receptor; i.p., intraperitoneal; P, postnatal day; s.c., subcutaneous.

3.2.2.1. Beneficial effects of caffeine

When the effects of caffeine on neurons were assessed in rat pups, high-dose
caffeine citrate (50 mg/kg/day) administration from postnatal day 1 (P1) to P12
increased total dendritic length and arborization of layer III pyramidal neurons
of the prefrontal cortex at P35 and this effect persisted after puberty (P70)
(Juarez-Mendez et al., 2006). The authors suggested that such changes in
dendritic arborization caused by caffeine could explain the improvement in
cognitive function reported in both children and rats (Bernstein et al., 1994;
Elkins et al., 1981; Prediger et al., 2005; Rapoport et al., 1981). A lower dose
of caffeine citrate (15–20 mg/kg/day) administered to rats from P2 to P6 has
been shown to reduce seizure susceptibility to some chemo-convulsants in
both juvenile and adult rats (Guillet and Dunham, 1995).
3.2.2.2. Neuroprotective effects of caffeine
It is evident from studies using animal models of perinatal brain injury that
caffeine may be neuroprotective; for example, in hypoxic-ischaemic rat pups
exposed from P0 to P12 to caffeine via lactating dams that were provided
water containing caffeine citrate (300 mg/L), myelination was enhanced and
ventriculomegaly reduced compared to untreated pups (Back et al., 2006).
Another study, using a similar method to the study above, showed that low-
dose caffeine administered to hypoxic-ischaemic rat pups via lactating dams
(300 mg/L) during the first week after birth (P1 to P7), significantly reduced
hypoxic-ischaemic damage to the ipsilateral cerebral hemisphere via a
reduction in neuronal necrosis and infarction (Bona et al., 1995). Reduced
ipsilateral cortical volume in hypoxic-ischaemic rats (induced at P7) treated
with saline was associated with significant deficits in spatial memory;
however, a single dose of caffeine citrate (10 mg/kg; i.p) administered
immediately following the induction of hypoxia-ischaemia, partly restored
cortical volume and attenuated deficits in spatial memory (Alexander et al.,
2013). These studies suggest both structural and functional benefits of
caffeine therapy in the setting of hypoxic-ischaemic perinatal brain injury;
whether caffeine has similar neuroprotective effects following brain injury
induced via fetal inflammation, intrauterine growth restriction, or preterm birth
is not known, nor is the optimum caffeine dose required to elicit these positive
effects.
The neuroprotective benefits of caffeine have been attributed to its effects on
Ca2+/cAMP response element binding protein (CREB) which mediates the
transcription of genes essential for the development and function of neurons;
such gene products include BDNF (Connolly and Kingsbury, 2010), a growth
factor involved in neuronal survival and in the maturation of developing
neurons (Connolly and Kingsbury, 2010; Cunha et al., 2010). It has been
shown that transcripts derived from endogenous CREB target genes, such as
the gene encoding BDNF, are increased in cell cultures following exposure to
clinically relevant concentrations of caffeine citrate (100–200 μM) (Connolly
and Kingsbury, 2010). It has been proposed that the observed increase in
BDNF levels as a result of caffeine treatment may contribute to the
neurological benefits that have been observed in infants receiving caffeine
(Connolly and Kingsbury, 2010); however, this has not yet been confirmed.
3.2.2.3. Detrimental effects of caffeine
A number of studies have shown that exposing the developing brain to
caffeine can adversely affect brain structure and function. One such study
showed that a caffeine-supplemented protein diet (20% protein + 1 mg/100 g
body weight caffeine) fed to rats from the day of birth (P0) to P43 increased
RNA and protein content within the cerebellum, but reduced the weight, DNA
content and RNA content of the whole brain at P43 (Yazdani et al., 1988). The
same authors later reported that a higher dose of caffeine (4 mg/100 g
maternal body weight) administered to newborn rats (via lactating dams) from
P0 to P10 reduced cerebellar weight and increased saturated fatty acid
concentration in the cerebellum when compared with untreated newborn rats
(Yazdani et al., 2004), indicating that caffeine alters de novo lipogenesis in the
brain. The authors argue that caffeine-induced changes in lipogenesis in the
cerebellum are unlikely to impact on myelin formation as myelination in rats
begins after the period of caffeine exposure (P10–P15); however, this was not
investigated. However, others have shown that caffeine reduces
oligodendroglial proliferation in vitro (Marret et al., 1993), and that neonatal
caffeine treatment (40 mg/kg and 80 mg/kg caffeine base) in rat pups (P2 to
P17 or P2 to P20) delays cerebral myelin synthesis (Fuller et al., 1982; Fuller
and Wiggins, 1981); thus further examination of the effects of caffeine on
cerebellar myelination is warranted.
Another study has shown a reduction in cell proliferation in the subventricular
zone and dentate gyrus in mice at P7 following daily administration of caffeine
base (10 mg/kg loading dose; 2.5 mg/kg maintenance dose) from P3 to P7
(Desfrere et al., 2007). This same study also found a decrease in the
formation of astrocytes in a number of brain regions including the cerebral
cortex and white matter; the authors suggest that this caffeine-induced
reduction in astrocyte formation is linked to the A2AAR antagonist properties of
caffeine, as these results could be mimicked using an A2AAR antagonist
(Desfrere et al., 2007). In addition to adversely affecting cell proliferation,
caffeine exposure during the early postnatal period increases apoptosis.
Specifically, a study in newborn rats showed that acute high-dose caffeine
administration (3 doses, 50 mg/kg; caffeine base) induced apoptosis
throughout the cerebral hemispheres via caspase-3-dependent mechanisms
(Kang et al., 2002). Similarly, administration of acute high-dose caffeine
(100 mg/kg; caffeine base) at P3 in rats resulted in an increase in apoptosis in
various brain regions including the cerebral cortex and the caudate nucleus
(Black et al., 2008).
Neonatal caffeine exposure also alters the level of A1AR binding and
ontogeny of A1ARs throughout the rodent brain, assessed using the specific
A1AR agonist [3H]cyclohexyladenosine (Etzel and Guillet, 1994; Guillet and
Kellogg, 1991). In comparison to saline treatment, administration of caffeine
citrate (20 mg/kg loading dose; 15 mg/kg maintenance dose) to newborn rats
from P2 to P6 increased A1AR binding in the cerebellum, cortex and
hippocampus across a range of postnatal ages (Guillet and Kellogg, 1991);
this led to the attainment of adult densities of A1ARs at earlier ages,
suggestive of accelerated development of A1ARs. In direct opposition to these
findings however, the authors subsequently reported a reduction in A1AR
binding in the molecular layer of the cerebellum (Etzel and Guillet, 1994) after
giving the same dose of caffeine citrate at the same postnatal ages as
described previously (Guillet and Kellogg, 1991); they suggest that the
difference between their studies was likely due to the limited postnatal period
over which the study was performed. More recently, caffeine treatment using
the same experimental protocol described above (20 mg/kg caffeine citrate
loading dose; 15 mg/kg maintenance dose; P2 to P6 in rats) (Etzel and
Guillet, 1994; Guillet and Kellogg, 1991) was associated with increased A1AR
and A2AAR labelling in several cardio-respiratory regions of the brain including
the hypothalamus and medulla (Gaytan and Pasaro, 2012). Despite the
conflicting data, it is conceivable that any divergence from the normal
development of A1ARs could lead to impaired brain function in the long term.
In support of this, caffeine exposure during development has been shown to
interfere with cholinergic neurotransmission in the brain and cause
behavioural deficits such as impaired learning in rats (da Silva et al., 2008;
Pan and Chen, 2007; Zimmerberg et al., 1991). Additionally, low-dose, acute
maternal caffeine exposure at 130 days of gestation in the ovine fetus
decreased cerebral oxygenation without any effects on fetal systemic
oxygenation (Tomimatsu et al., 2007).
3.2.2.4. No adverse effect
To date one study suggests that high-dose caffeine has no detrimental effect
on the developing brain. Specifically, chronic daily high-dose caffeine
administration (25 mg/kg loading dose; 20 mg/kg maintenance dose; caffeine
base) in fetal sheep from 0.7 to 0.8 of term (a stage when white matter
development is similar to that of preterm infants born at about 27–34 weeks
gestational age) did not affect glial cell density (astrocytes, microglia or
oligodendrocytes), myelination or neuronal cell density in the developing
cerebral white or grey matter (Atik et al., 2014). Whether or not high-dose
caffeine affects other brain regions including the cerebellum, or has a longer-
term impact on brain structure still needs to be assessed.
4. Conclusions
The studies reviewed here relating to the effects of caffeine on
neurodevelopment provide substantial evidence that caffeine can have both
beneficial and detrimental consequences for the immature brain; the effects of
caffeine are apparently dependent on the species examined, the dose of
caffeine administered, the stage of neurodevelopment at the time of
administration and the duration of exposure. While moderate doses of caffeine
appear to be clinically beneficial for preterm infants, assessment of the cellular
and molecular effects of caffeine on the developing brain using animal models
suggests otherwise, with most studies suggesting that caffeine has adverse
effects on the developing brain regardless of the dose administered. However,
a limitation of these animal studies is that they do not consider the interaction
between caffeine and co-therapies such as positive pressure ventilation and
oxygen therapy, which on their own are associated with adverse
neurodevelopmental outcomes in infants. Additionally, many of these studies
have not been conducted in clinically relevant animal models; thus, in future,
greater attention must be placed on assessing the impact of caffeine at a
range of doses on the structure and function of the developing brain in
preclinical studies, especially in clinically relevant animal models. As caffeine
has been, and will likely remain, the treatment of choice for AOP, infants who
do not respond to standard doses of caffeine may receive higher doses.
Therefore future studies must focus on determining the maximal dose of
caffeine that is not only effective for AOP, but is also safe for the developing
brain of preterm infants.

Funding
The authors are supported by the National Health and Medical Research
Council (NHMRC) of Australia (MT, JC, RDM, LD and RH, grant ID# 628312)
and the Victorian Government’s Operational Infrastructure Support Scheme.
JC is an NHMRC Early Career Fellow (ID# 1053787) and MT is an RMIT Vice
Chancellor’s Senior Research Fellow.