1. 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 C’s 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. In Search of a Clinically Relevant Tapering Protocol for Hydrocortisone Treatment in Newborn Animals Lee Y, Huang LD, Vázquez DM Department of Pediatrics University of Michigan, Ann Arbor, MI Research Supported by National Institute of Mental Health RO1 HD/DK37431 Abstract 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. Objectives & Hypothesis Results References Conclusions 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. Methods 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 (1:20 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 < 0.05 was considered significant. 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-cognitive 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. 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. 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. * p<0.05 compared to VEH, # p<0.05 compared to HC. 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. * p<0.05 compared to VEH. 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. * p<0.05 compared to 0 dose (VEH). All Dexamethasone Protocols increased Surfactant Transcription in Lung Tissue Four Day and Late 2 Day Dexamethasone Protocol increased Surfactant Protein Expression in Lung We determined a hydrocortisone dose that was effective in increasing SP-A protein level in lung. VEH DEX HC 3 4 5 6 7 Protocol C Protocol B Post-natal Days (PD) kill spleen lungs Tissue Collected PTC Dexamethasone (DEX, mg/kg BW) Hydrocortisone (HC, mg/kg BW) PD3PD4PD5PD6PD3PD4PD5PD6 A0.50.250.1250.055.02.01.00.5 B 0.15.01.0 C0.50.15.01.0 Table1: Postnatal (PD) Time and Dosing Schedules for Protocols (PTC) –A, –B & -C Protocol A PD Fig 1. SP-A Western Blotting. 31-35 KD Spleen lung lung SP-A (-) ctrl male female (+) ctrl Fig 1. SP-A Western Blotting 31-35 KD