In Search of a Clinically Relevant Tapering Protocol for Hydrocortisone Treatment in Newborn Animals Lee Y, Huang LD, V

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In Search of a Clinically Relevant Tapering
Protocol for Hydrocortisone Treatment in Newborn
Animals Lee Y, Huang LD, Vázquez DMDepartment
of PediatricsUniversity of Michigan, Ann Arbor,
MI
Abstract
Results
Conclusions

Glucocorticoid (GC) steroid hormone is currently
used to treat premature infants by promoting the
production of surfactant proteins that prevent
air exchange saccules from collapsing. Studies in
animal models concur that Dexamethasone (DEX)
enhances lung development and surfactant protein
(SP) A, -B and -C production. However, chronic
postnatal DEX treatment leads to severe
side-effects, such as cerebral palsy. Thus, we
compared DEX with a less potent GC,
hydrocortisone (HC) to explore HC doses that
would be clinically effective. Using an animal
model we compared and measured lung SP-A level
following three different DEX and HC dosing
schedules. Dosing protocol A consisted of four
injections of decreasing dosage of DEX or HC, on
days 3, 4, 5, 6 after birth protocol B
injections were given on days 3 and 4 whereas,
protocol Cs injections were given on days 5 and
6. Saline treated animals served as controls.
SP-A gene and protein expression was determined
by real time PCR and Western blotting,
respectively. The DEX-treated group had
significant SP-A protein production as expected,
and this was observed when the pups were treated
throughout the 4 days (days 3-6) or on the last 2
days of the protocol (days 5 6). Therefore,
protocol C (dosing on days 5 6) was chosen to
further determine an HC dose that would be
effective at promoting SP-A synthesis. We
determined a tapering dose regimen for HC that
effectively increased SP-A protein in lung.
Dexamethasone and hydrocortisone are effective
steroids that increase surfactant protein in
lung, therefore improving lung function (Jones,
1977). Dexamethasone was prevalently used to
treat premature infants until it was substituted
for its lethal side effects, including cerebral
palsy and developmental delay (Shinwell et al.,
2000). Chronic dexamethasone treatments may
permanently damage brain, neurological
development, altering infants behavior,
cognition and learning abilities. While steps
have been taken to eliminate the clinical use of
dexamethasone in the premature human population,
its use has been replaced by the less potent
hydrocortisone steroid equivalent. It is unclear
if hydrocortisone use will result in similar, yet
more subtle neuro-behavioral-cognitive effects as
dexamethasone. Our results show that we can
apply a dose regimen that is clinically relevant.
SP-A protein level increased when the starting
hydrocortisone dose are 5 and 20 mg/kg BW. A
decreasing pattern of SP-A synthesis from
starting dose of 80mg/kg BW may be due to an
effect of down regulation of the glucocorticoid
receptor with this higher dose (Islam and
Mendelson, 2008). We can now utilize an optimal
hydrocortisone dose to explore neuro-behavioral-co
gnitive effects in an animal model. We predict
that the results of such studies will be crucial
to inform clinical management of critically ill
infants with respiratory problems.
All Dexamethasone Protocols increased Surfactant
Transcription in Lung Tissue

Figure 1. SP-A mRNA Levels in Lung Protocols
A, B and C. No sex effect demonstrated.
Dexamethasone had equally significant results
with all drug dosing protocols. no protocol
differences. plt0.05 compared to VEH,
plt0.05 compared to HC.
Objectives Hypothesis
Four Day and Late 2 Day Dexamethasone Protocol
increased Surfactant Protein Expression in Lung
The aim of the project is to search for a
clinically relevant tapering protocol for
hydrocortisone treatment in newborn animals.
Hypotheses a) Dexamethasone will induce
surfactant synthesis regardless of the dose and
post-natal time it is used b) Current
hydrocortisone doses used in our animal model
will not induce surfactant synthesis in lung
tissue.
Methods
1. Animal Procedure Pregnant Sprague-Dawley rats
of known gestational age were housed individually
according to NIH guidelines until the day of
birth, postnatal day 1 (PD1). On PD2, each litter
was culled to 6 males and 6 females. Three groups
were treated with 3 different protocols. Tissue
was collected 24 hr after the last treatment day.
weighed and immediately stored frozen at -80?C.
References
Table1 Postnatal (PD) Time and Dosing Schedules
for Protocols (PTC) A, B -C
Figure 2. SP-A Protein Level in Lung Protocols
A, B and C. There was no sex effect
demonstrated. SP-A protein level was
significantly elevated in lung tissue with Dex
following Protocols A and C. Recall that
Protocol A injected four days starting form PND
3, but protocol C injected only two days starting
from PND 5. Protocol C was selected to further
investigate the optimal HC dosage. plt0.05
compared to VEH.
Halliday, H. L., Ehrenkranz, R. A., 2001.
Moderately early (7-14 days) postnatal
corticosteroids for preventing chronic lung
disease in preterm infants. Cochrane Database
Syst Rev. CD001144. Islam, K. N., Mendelson, C.
R., 2008 Glucocorticoid receptor inhibition of
surfactant protein-A (SP-A) gene expression in
lung type II cells is mediated by repressive
changes in histone modification at the SP-A
promoter. Mol Endocrinol. 22, 585-96. Jones, M.
B., 1977. Respiratory distress syndrome and the
induction of fetal lung maturity by the use of
glucocorticoids. JOGN Nurs. 6, 21-8. Shinwell,
E. S., et al., 2000. Early postnatal
dexamethasone treatment and increased incidence
of cerebral palsy. Arch Dis Child Fetal Neonatal
Ed. 83, F177-81. Watterberg, K. L., et al.,
1997. Chorioamnionitis, cortisol, and acute lung
disease in very low birth weight infants.
Pediatrics. 99, E6.
PTC Dexamethasone (DEX, mg/kg BW) Dexamethasone (DEX, mg/kg BW) Dexamethasone (DEX, mg/kg BW) Dexamethasone (DEX, mg/kg BW) Hydrocortisone (HC, mg/kg BW) Hydrocortisone (HC, mg/kg BW) Hydrocortisone (HC, mg/kg BW) Hydrocortisone (HC, mg/kg BW)
PD3 PD4 PD5 PD6 PD3 PD4 PD5 PD6
A 0.5 0.25 0.125 0.05 5.0 2.0 1.0 0.5
B 0.5 0.1 5.0 1.0
C 0.5 0.1 5.0 1.0
PD
Protocol A
We determined a hydrocortisone dose that was
effective in increasing SP-A protein level in
lung.
2. Western Blot Analysis for Surfactant Protein A
(SP-A) Right side lung and spleen were
homogenized in lysis buffer, incubated on ice,
and centrifuged. Western blot analysis was
performed using 20ug of total protein. The SP-A
Primary antibody and goat anti-rabbit horseradish
peroxidase (2nd antibody) were conjugated and
detected by ECL reagent. Spleen served as a
negative control. 3. RNA Isolation, cDNA
synthesis and Real-time PCR of SP-A Using left
side lung, reactions were carried out in a
Bio-Rad iCycler. PCR reactions (50ul) contained
10ul of cDNA template (120 dilution) and 40ul of
master mix. After PCR is finished, the PCR
specificity was examined by 2 agarose gel. 4.
Data Analysis In all cases, rats were identified
by number only. ANOVA with p value lt 0.05 was
considered significant.
Research Supported by
National Institute of Mental Health RO1 HD/DK37431
Figure 3. HC Dose Response Curve Optimal HC
Dosing Protocol. Four starting HC doses were
investigated (5, 20, 80 and 160 mg/kg of body
weight (BW). HC dose of 5 and 20 mg/kg BW were
significantly effective in increasing SP-A
protein level compared to VEH. plt0.05 compared
to 0 dose (VEH).