Effects of antenatal inflammation and postnatal oxygen fluctuation on developing white matter in a rodent model of prematurity
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Date
29/11/2016Author
Pilley, Elizabeth Sarah
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Abstract
Inflammation and oxidative stress are increasingly recognised as important
independent mediators of preterm brain injury and have been implicated in the
pathogenesis of cerebral palsy and cognitive impairment. Such exposures are
common for the premature infant in whom infection and inflammatory morbidities
occur in around 60%. Furthermore, many preterm infants require oxygen therapy
and respiratory support due to lung immaturity. Epidemiological and experimental
studies indicate that in addition to the independent effects of inflammation and
extreme hyperoxia on the developing brain, inflammation preconditions the
developing brain resulting in variable injury when exposed to subsequent hypoxia-ischaemia.
However experimental studies employing exposure to more modest
oxygen fluctuations are lacking. This thesis characterises a clinically relevant model
of prematurity where the developing brain is exposed to low grade inflammation and
oxygen fluctuation around a hyperoxic mean. We hypothesise that antenatal
inflammation and postnatal oxygen fluctuation, both alone and in combination, have
detrimental effects on developing white matter.
Pregnant dams received intraperitoneal lipopolysaccharide (LPS) or saline on G18
and G19. Dams and their pups were then reared in room air or fluctuating hyperoxia
(circa 10kPa) for seven days. We measured longitudinal brain and body growth in
different experimental groups to 12 weeks. Whole brains were examined for mRNA
expression of inflammatory cytokines (TNFα, IL-1β, IL-6 and IL-10) and markers of
oxidative injury (iNOS, SOD2). To determine the effect of perinatal insults on
developing white matter, we analysed the expression of myelin basic protein (MBP)
and glial fibrillary acidic protein (GFAP) in the internal and external capsule. We
also examined white matter tracts for differences in microglia (CD68),
oligodendrocyte progenitor cells (NG2), oligodendroglial cells (Olig2) and cell death
(cleaved caspase3). Behavioural studies (Morris Watermaze Test, Elevated Plus Test
and Open Field Test) were undertaken at 12 weeks of age to detect any long-term
functional difference between the groups.
Antenatal inflammation reduces both brain and body growth at P7. This normalises
by P14 unless this inflammatory insult has been followed by postnatal oxygen
fluctuation, where brain and body growth restriction persists until P14. We defined
our inflammatory response at P1 following antenatal inflammation and did not
observe elevation of mRNA at P1. We demonstrated increased SOD2 at this time
point, indicating a reparative process. At P7 we observed a significant reduction in
the oxidative response following combined exposure to antenatal inflammation and
postnatal oxygen fluctuation, indicating a potential limit to, or suppression of, the
reparative process.
In terms of white matter injury, antenatal inflammation reduces myelination at P7.
There is no synergistic effect of inflammation and oxygen fluctuation on MBP
immunohistochemistry at P7. However, MBP mRNA expression is increased in
pups exposed to both insults compared to those exposed to inflammation alone
suggesting that the oxygen fluctuation may stimulate MBP production in response to
oxidative injury. MBP mRNA levels and protein expression have all normalised by
P14. We observed a reduction in total cell number in the external capsule and corpus
callosum in the dual insult group, without an increase in caspase. In keeping with
other studies we detected no effect of our perinatal insults on NG2+ve
oligodendrocytes. Olig2+ve cell numbers were also consistent between experimental
groups.
In further characterisation of the cellular response, antenatal inflammation followed
by postnatal oxygen fluctuation resulted in a decrease in GFAP mRNA at P7, an
effect which was reversed and significantly increased by P14 suggesting delayed
activation of the innate immune system. No difference was observed in microglial
numbers between experimental groups. There was however, increased microglial
cell death (CD68 + caspase) in the group exposed to antenatal inflammation. When
this insult was combined with postnatal oxygen fluctuation there was a comparative
decrease in microglial cell death, which may reflect an earlier peak of microglial cell
death, due to an increased and sustained inflammatory stimulus. Morris Watermaze testing demonstrated that pups exposed to both insults took longer
than controls to locate the hidden platform on day 1, which is a measure of spatial
learning. The Elevated Plus Test and Open Field Test demonstrated that pups
exposed to both insults were less anxious and took more risks than pups exposed to
single insults.
In conclusion, within a clinically relevant preterm model, antenatal inflammation
transiently disrupts both brain and body growth and myelination of the motor tracts
of the developing brain. Moreover, when combined with postnatal oxygen
fluctuation, detrimental effects on growth are amplified and sustained. Decreased
cell numbers are also observed within white matter tracts. In terms of long term
functionality, these pups display disinhibition of behaviour as young adults.
Collectively, this thesis demonstrates that synergistic actions of common low-grade
perinatal insults may alter normal neurodevelopment, and that this may carry a risk
of neurodevelopmental sequelae for preterm infants.
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