Summer Research Internships Prepare High School Students for 21st Century Biomedical Careers.

STEM internships for both high school and college students provide early opportunities for students to discover careers of interest and career paths they may not otherwise experience. For over 25 years, the University of Alabama at Birmingham's (UAB) Center for Community OutReach Development (CORD) has provided rising high school seniors with opportunities to conduct research in federally-funded laboratories under the mentorship of UAB faculty. This paper evaluates CORD's High School Summer Science Institute III Program (SSI III) and its impact on participants' STEM career trajectories. Outcomes were tracked for SSI III participants over an eight-year period, and former interns' perceptions of the program reported. Over 99% of surveyed interns (N=102) chose a STEM undergraduate major, and 97% of the former interns reported they were pursuing STEM careers. Nearly all interns indicated their SSI-III experience was very positive and influenced their career decision. Over half of the interns matriculated into an undergraduate STEM major at UAB, providing the university with return as more excellent students for their investment in the program. These results highlight the importance of high school student involvement in STEM internships as a pathway that leads towards STEM careers.

Neurodevelopmental impairment following neonatal hyperoxia in the mouse.

Extremely premature infants are often exposed to supra-physiologic concentrations of oxygen, and frequently have hypoxemic episodes. These preterm infants are at high risk (~40%) for neurodevelopmental impairment (NDI) even in the absence of obvious intracranial pathology such as intraventricular hemorrhage or periventricular leukomalacia. The etiology for NDI has not been determined, and there are no animal models to simulate neurodevelopmental outcomes of prematurity. Our objectives were to develop and characterize a mouse model to determine long-term effects of chronic hypoxia or hyperoxia exposure on neurodevelopment. Newborn C57BL/6 mice were exposed to hypoxia (12% O(2)) or hyperoxia (85% O(2)) from postnatal days 1 to 14 and then returned to air. At 12-14 weeks of age, neurobehavioral assessment (Water Maze test, Novel Object Recognition test, Open Field test, Elevated Plus Maze, and Rotarod test) was performed, followed by MRI and brain histology. Neurobehavioral testing revealed that hyperoxia-exposed mice did poorly on the water maze and novel object recognition tests compared to air-exposed mice. MRI demonstrated smaller hippocampi in hyperoxia- and hypoxia-exposed mice with a greater reduction in hyperoxia-exposed mice, including a smaller cerebellum in hyperoxia-exposed mice. Brain histology showed reduced CA1 and CA3 and increased dentate gyral width in hippocampus. In conclusion, neonatal hyperoxia in mice leads to abnormal neurobehavior, primarily deficits in spatial and recognition memory, associated with smaller hippocampal sizes, similar to findings in ex-preterm infants. This animal model may be useful to determine mechanisms underlying developmental programming of NDI in preterm infants, and for evaluation of therapeutic strategies.

Titanium oxide nanoparticle instillation induces inflammation and inhibits lung development in mice.

Nanoparticles are used in an increasing number of biomedical, industrial, and food applications, but their safety profiles in developing organisms, including the human fetus and infant, have not been evaluated. Titanium oxide (TiO(2)) nanoparticles, which are commonly used in cosmetics, sunscreens, paints, and food, have been shown to induce emphysema and lung inflammation in adult mice. We hypothesized that exposure of newborn mice to TiO(2) would induce lung inflammation and inhibit lung development. C57BL/6 mice were exposed to TiO(2) (anatase; 8-10 nm) nanoparticles by intranasal instillation as a single dose on postnatal day 4 (P4) or as three doses on postnatal days 4, 7, and 10 (each dose = 1 µg/g body wt). Measurements of lung function (compliance and resistance), development (morphometry), inflammation (histology; multiplex analysis of bronchoalveolar lavage fluid for cytokines; PCR array and multiplex analysis of lung homogenates for cytokines) was performed on postnatal day 14. It was observed that a single dose of TiO(2) nanoparticles led to inflammatory cell influx, and multiple doses led to increased inflammation and inhibition of lung development without significant effects on lung function. Macrophages were noted to take up the TiO(2) nanoparticles, followed by polymorphonuclear infiltrate. Multiple cytokines and matrix metalloproteinase-9 were increased in lung homogenates, and VEGF was reduced. These results suggest that exposure of the developing lung to nanoparticles may lead to ineffective clearance by macrophages and persistent inflammation with resulting effects on lung development and may possibly impact the risk of respiratory disorders in later life.

Transforming growth factor-ß regulates endothelin-1 signaling in the newborn mouse lung during hypoxia exposure.

We have previously shown that inhibition of transforming growth factor-ß (TGF-ß) signaling attenuates hypoxia-induced inhibition of alveolar development and abnormal pulmonary vascular remodeling in the newborn mice and that endothelin-A receptor (ETAR) antagonists prevent and reverse the vascular remodeling. The current study tested the hypothesis that inhibition of TGF-ß signaling attenuates endothelin-1 (ET-1) expression and thereby reduces effects of hypoxia on the newborn lung. C57BL/6 mice were exposed from birth to 2 wk of age to either air or hypoxia (12% O(2)) while being given either BQ610 (ETAR antagonist), BQ788 (ETBR antagonist), 1D11 (TGF-ß neutralizing antibody), or vehicle. Lung function and development and TGF-ß and ET-1 synthesis were assessed. Hypoxia inhibited alveolar development, decreased lung compliance, and increased lung resistance. These effects were associated with increased TGF-ß synthesis and signaling and increased ET-1 synthesis. BQ610 (but not BQ788) improved lung function, without altering alveolar development or increased TGF-ß signaling in hypoxia-exposed animals. Inhibition of TGF-ß signaling reduced ET-1 in vivo, which was confirmed in vitro in mouse pulmonary endothelial, fibroblast, and epithelial cells. ETAR blockade improves function but not development of the hypoxic newborn lung. Reduction of ET-1 via inhibition of TGF-ß signaling indicates that TGF-ß is upstream of ET-1 during hypoxia-induced signaling in the newborn lung.

Hypoxia-induced inhibition of lung development is attenuated by the peroxisome proliferator-activated receptor-γ agonist rosiglitazone.

Hypoxia enhances transforming growth factor-ß (TGF-ß) signaling, inhibiting alveolar development and causing abnormal pulmonary arterial remodeling in the newborn lung. We hypothesized that, during chronic hypoxia, reduced peroxisome proliferator-activated receptor-γ (PPAR-γ) signaling may contribute to, or be caused by, excessive TGF-ß signaling. To determine whether PPAR-γ was reduced during hypoxia, C57BL/6 mice were exposed to hypoxia from birth to 2 wk and evaluated for PPAR-γ mRNA and protein. To determine whether rosiglitazone (RGZ, a PPAR-γ agonist) supplementation attenuated the effects of hypoxia, mice were exposed to air or hypoxia from birth to 2 wk in combination with either RGZ or vehicle, and measurements of lung histology, function, parameters related to TGF-ß signaling, and collagen content were made. To determine whether excessive TGF-ß signaling reduced PPAR-γ, mice were exposed to air or hypoxia from birth to 2 wk in combination with either TGF-ß-neutralizing antibody or vehicle, and PPAR-γ signaling was evaluated. We observed that hypoxia reduced PPAR-γ mRNA and protein, in association with impaired alveolarization, increased TGF-ß signaling, reduced lung compliance, and increased collagen. RGZ increased PPAR-γ signaling, with improved lung development and compliance in association with reduced collagen and TGF-ß signaling. However, no reduction was noted in hypoxia-induced pulmonary vascular remodeling. Inhibition of hypoxia-enhanced TGF-ß signaling increased PPAR-γ signaling. These results suggest that hypoxia-induced inhibition of lung development is associated with a mutually antagonistic relationship between reduced PPAR-γ and increased TGF-ß signaling. PPAR-γ agonists may be of potential therapeutic significance in attenuating TGF-ß signaling and improving alveolar development.

Vitamin A and retinoic acid act synergistically to increase lung retinyl esters during normoxia and reduce hyperoxic lung injury in newborn mice.

We have shown that vitamin A (VA) and retinoic acid (RA) synergistically increase lung retinyl ester content in neonatal rats. To confirm whether this biochemical synergism attenuates early neonatal hyperoxic lung injury in mice, we exposed newborn C57BL/6 mice to 95% O2 or air from birth to 4 d. The agent [vehicle, VA, RA, or the combination vitamin A+retinoic acid (VARA)] was given orally daily. Lung and liver retinyl ester content was measured, and lung injury and development were evaluated. We observed that lung, but not liver, retinyl ester levels were increased more by VARA than by VA or RA alone. Hyperoxic lung injury was reduced by VA and RA, and more so by VARA. VARA attenuated the hyperoxia-induced increases in macrophage inflammatory protein (MIP)-2 mRNA and protein expression, but did not alter hyperoxia-induced effects on peptide growth factors (PDGF, VEGF, and TGF-beta1). The 4-d exposure to hyperoxia or retinoids did not lead to observable differences in lung development. We conclude that the VARA combination has synergistic effects on lung retinyl ester concentrations and on the attenuation of hyperoxia-induced lung injury in newborn mice, possibly by modulation of inflammatory mediators.

Evaluation of a pumpless lung assist device in hypoxia-induced pulmonary hypertension in juvenile piglets.

Persistent pulmonary hypertension is an important cause of mortality and morbidity in term infants. The lung assist device (LAD) is a novel, pumpless, low-resistance extracorporeal oxygenator to supplement mechanical ventilation. The LAD may be associated with fewer complications compared with conventional extracorporeal membrane oxygenation. The objective was to test the feasibility and efficacy of the LAD in juvenile piglets with hypoxia-induced pulmonary hypertension. Pulmonary hypertension was acutely induced by hypoxia in six 3- to 4-wk-old acutely instrumented and intubated piglets. The LAD was attached between a carotid artery and jugular vein. Gas exchange and hemodynamic variables, including pulmonary arterial pressure (PAP) and cardiac output (CO), were measured. Successful LAD cannulation was achieved without complications in all animals. Extracorporeal shunt flow through the device averaged 18% of CO. The LAD achieved oxygen delivery of 20% of total oxygen consumption. PAP was reduced by 35% from 28 +/- 5 to 18 +/- 4 mm Hg (p < 0.05) and systemic Pao2 increased by 33% from 27 +/- 2 to 36 +/- 4 mm Hg (p < 0.05). Other hemodynamic variables remained stable. The novel LAD shows feasibility and efficacy in improving gas exchange and reducing PAPs in a juvenile animal model of hypoxia-induced pulmonary hypertension.

A pumpless lung assist device reduces mechanical ventilation-induced lung injury in juvenile piglets.

Respiratory failure is a major contributor to mortality and morbidity in newborn infants. The lung assist device (LAD) is a novel gas exchange device that supplements mechanical ventilation. The objective is to test the effect of the LAD on pulmonary histopathology in juvenile piglets with acute lung injury caused by saline lung lavage (SLL) followed by intermittent mandatory ventilation (IMV). Three- to 4-wk-old piglets were randomized to no intervention (control group), SLL alone (SLL group), SLL + IMV (IMV group), or SLL + IMV + LAD (LAD group) (n = 6 per group). The carotid artery and jugular vein were cannulated and an arteriovenous circuit completed, and the LAD was inserted into this circuit. Gas exchange via the LAD was initiated by passage of 100% oxygen over the blood-carrying hollow fibers of the LAD. Hemodynamic variables were recorded. Mechanical ventilation was systematically weaned. Lung histology was scored by two observers masked to treatment group. There were no differences in hemodynamic variables between the study groups. There was a significant increase in the total lung injury score in the IMV group compared with the LAD group. The novel pumpless low-resistance LAD has shown feasibility and potential to decrease ventilator-induced lung injury in a juvenile animal model.

Loss of Thy-1 inhibits alveolar development in the newborn mouse lung.

Transforming growth factor (TGF)-beta mediates hypoxia-induced inhibition of alveolar development in the newborn lung. TGF-beta is regulated primarily at the level of activation of latent TGF-beta. Fibroblasts expressing Thy-1 (CD90) inhibit TGF-beta activation. We hypothesized that loss of Thy-1 due to hypoxia may be a mechanism by which hypoxia increases TGF-beta activation and that animals deficient in Thy-1 will simulate the effects of hypoxia on lung development. To determine if loss of Thy-1 occurred during hypoxia, non-transgenic (C57BL/6) wild-type (WT) mice exposed to hypoxia were evaluated for Thy-1 mRNA and protein. To determine if Thy-1 deficiency simulated hypoxia, WT and Thy-1 null (Thy-1(-/-)) mice were exposed to air or hypoxia from birth to 2 wk, the critical period of lung development, and lung histology, function, parameters related to TGF-beta signaling, and extracellular matrix protein content were measured. To test if the phenotype in Thy-1(-/-) mice was due to excessive TGF-beta signaling, measurements were also performed in Thy-1(-/-) mice administered TGF-beta neutralizing antibody (1D11). We observed that hypoxia reduced Thy-1 mRNA and Thy-1 staining in WT mice. Thy-1(-/-) mice had impaired alveolarization, increased TGF-beta signaling, reduced lung epithelial and endothelial cell proliferation but increased fibroblast proliferation, and increased collagen and elastin. Lung compliance was lower, and tissue but not airway resistance was higher in Thy-1(-/-) mice at 2 wk. Thy-1(-/-) mice given 1D11 had improved alveolar development and lung function. These data support the hypothesis that hypoxia, by reducing Thy-1, increases TGF-beta activation, and thereby inhibits normal alveolar development.

Transforming growth factor-beta signaling mediates hypoxia-induced pulmonary arterial remodeling and inhibition of alveolar development in newborn mouse lung.

Hypoxia causes abnormal neonatal pulmonary artery remodeling (PAR) and inhibition of alveolar development (IAD). Transforming growth factor (TGF)-beta is an important regulator of lung development and repair from injury. We tested the hypothesis that inhibition of TGF-beta signaling attenuates hypoxia-induced PAR and IAD. Mice with an inducible dominant-negative mutation of the TGF-beta type II receptor (DNTGFbetaRII) and nontransgenic wild-type (WT) mice were exposed to hypoxia (12% O(2)) or air from birth to 14 days of age. Expression of DNTGFbetaRII was induced by 20 microg/g ZnSO(4) given intraperitoneally daily from birth. PAR, IAD, cell proliferation, and expression of extracellular matrix (ECM) proteins were assessed. In WT mice, hypoxia led to thicker, more muscularized resistance pulmonary arteries and impaired alveolarization, accompanied by increases in active TGF-beta and phosphorylated Smad2. Hypoxia-induced PAR and IAD were greatly attenuated in DNTGFbetaRII mice given ZnSO(4) compared with WT control mice and DNTGFbetaRII mice not given ZnSO(4). The stimulatory effects of hypoxic exposure on pulmonary arterial cell proliferation and lung ECM proteins were abrogated in DNTGFbetaRII mice given ZnSO(4). These data support the conclusion that TGF-beta plays an important role in hypoxia-induced pulmonary vascular adaptation and IAD in the newborn animal model.

Role of matrix metalloproteinase-2 in newborn mouse lungs under hypoxic conditions.

Hypoxia impairs normal neonatal pulmonary artery remodeling and alveolar development. Matrix metalloproteinase-2 (MMP-2), which regulates collagen breakdown, is important during development. Our objective was to test the hypothesis that hypoxia attenuates the normal postnatal increase in MMP-2 and evaluate alveolar development and pulmonary arterial remodeling in Mmp2 mice. C57BL/6 wild-type (WT), Mmp2, Mmp2, and MMP-inhibited (with doxycycline) mice were exposed to hypoxia (12% O2) or air from birth to 2 wk of age. Pulmonary arterial remodeling, alveolar development, and vascular collagen and elastin were evaluated. MMP-2 was estimated by quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, immunohistochemistry, and zymography. We observed that 1) in WT mice, hypoxia led to thicker-walled pulmonary arteries and impaired alveolarization, accompanied by decreased MMP-2 and increased tissue inhibitor of metalloproteinases-2 (TIMP-2); 2) Mmp2 mice in air had thicker-walled arteries, impaired alveolarization, and increased perivascular collagen and elastin compared with WT; 3) hypoxia further inhibited alveolarization but did not alter arterial thickening in Mmp2 mice. Mmp2 and MMP-inhibited mice also had thicker-walled arteries than WT in air, but alveolarization was not different. We conclude that hypoxia reduces the postnatal MMP-2 increase in the lung, which may contribute to abnormal pulmonary arterial remodeling and impaired alveolarization.

Endothelin-1 mediates hypoxia-induced increases in vascular collagen in the newborn mouse lung.

Endothelin-1 (ET-1) mediates hypoxia-mediated pulmonary vascular remodeling (HPVR), and endothelin-A receptor (ET-AR) blockade prevents HPVR in newborn mice. Our objective was to determine postnatal effects of chronic hypoxia and/or ET-AR blockade on lung ET-1, ET-AR, ET-BR, and vascular collagen and elastin. Newborn C57BL/6 mice (n = 6-8/gp) given either BQ610 (ET-AR blocker) or vehicle were exposed to air or hypoxia (12% O2) from birth for 1, 3, or 14 d. Lung ET-1 was assessed by ELISA, and ET-AR and ET-BR by immunohistochemistry. Vascular collagen and elastin were assessed by quantitative image analysis. ET-1, ET-AR, ET-BR, collagen I and III, and tropoelastin mRNA levels were assessed by real-time quantitative RT-PCR. We observed that: 1) hypoxia attenuated the normal postnatal decrease in ET-1 and collagen content; 2) ET-AR blockade reduced collagen independent of O2; 3) hypoxia increased elastin mRNA expression and attenuated the normal postnatal decrease in elastin content; and 4) BQ610 reduced elastin mRNA but not elastin content. We conclude that, in neonatal mice, hypoxia attenuates normal postnatal decreases in ET-1, vascular collagen, and elastin. ET-AR blockade reduces collagen fiber area but not mRNA, and does not decrease elastin despite reducing its expression.

Endothelin-A receptor blockade prevents and partially reverses neonatal hypoxic pulmonary vascular remodeling.

Hypoxia-induced pulmonary vascular remodeling (HPVR) may lead to persistent pulmonary hypertension of the newborn or cor pulmonale. Endothelin-1 (ET-1), via endothelin-A (ET(A)) receptor activation, mediates hypoxic pulmonary vasoconstriction. Our objectives were to develop a newborn mouse model of HPVR and to test the hypothesis that ET(A) blockade would prevent and reverse HPVR in this model. C57BL/6 mice (n = 64) were exposed to 12% oxygen (HYP group) or room air (RA group) from birth to 2 wk of age. The mice were injected intraperitoneally daily with either BQ-610 (ET(A) blocker) or vehicle (cottonseed oil) from birth (prevention study) or from 6 d of age (reversal study). HPVR was assessed histologically by pulmonary vascular morphometry by an examiner masked to study group, and by measurement of the right ventricle to left ventricle (RV/LV) thickness ratio. Hypoxia increased medial wall thickness (%WT) in pulmonary arteries <100 mum in diameter and RV/LV thickness ratio. BQ-610 prevented the hypoxia-induced increase in %WT and RV/LV thickness ratio when given from birth, and later therapy partially reversed the hypoxia-induced increase in %WT but not RV/LV thickness ratio. These data show that in the newborn mouse model, chronic hypoxia leads to HPVR that can be completely prevented and partially reversed by ET(A) blockade. These results indicate that ET-1, acting via ET(A) receptors, is a mechanism of pathophysiologic significance underlying neonatal HPVR. Development of this newborn mouse model of HPVR facilitates investigation of mechanisms underlying this important and severe disease entity in human infants.

Endothelin-A receptor blockade in porcine pulmonary hypertension.

Endothelin-1 can cause pulmonary vasoconstriction via endothelin-A (ET(A)) receptor activation. We hypothesized that ET(A) blockers (EMD 122946 and BQ 610) would reduce hypoxia-induced (HYP) but not group B streptococcal infusion (GBS)-induced pulmonary hypertension in a juvenile whole animal model. Pulmonary hypertension was created by exposing chronically instrumented piglets to HYP (n = 12) or heat-killed GBS (n = 11). ET(A) blockade was produced by increasing bolus doses of EMD122946 or BQ 610. Pulmonary arterial pressure (PAP), systemic arterial pressure (SAP), left atrial pressure, central venous pressure, and cardiac output were continuously measured. Pulmonary and systemic vascular resistance indices (PVRI and SVRI) were calculated. HYP doubled PAP and PVRI. Both ET(A) blockers decreased PAP and PVRI in a dose-dependent manner in HYP, with high doses decreasing PVRI to baseline and reducing PAP by 50%. GBS also doubled both PAP and PVRI. EMD 122946 did not change PAP or PVRI in GBS, although BQ 610 markedly increased PVRI (>100% increase with 0.15 mg/kg) and showed a trend toward increasing PAP. Both models showed minimal (<25%) changes in SAP or SVRI. Neither ETA blocker changed baseline hemodynamics in the absence of HYP or GBS. PaO(2) did not change with GBS but decreased with BQ 610. ET(A) receptor blockade attenuated hypoxic, but not GBS induced pulmonary hypertension. BQ 610 worsened PVRI and oxygenation in the GBS model. Differences in response to ET(A) blockade in pulmonary hypertension may be seen depending on the etiology (hypoxia versus infection-associated), and the specific ET(A) antagonist used.

Feasibility of nitric oxide administration by oxygen hood in neonatal pulmonary hypertension.

OBJECTIVE: To test the hemodynamic efficacy and feasibility of nitric oxide (NO) administration by oxygen hood in neonatal pulmonary hypertension. STUDY DESIGN: A double-hood apparatus was used in which a combination of NO, O(2), and N(2) was introduced into the inner hood and suctioned from the outer hood. Chronically instrumented non-intubated piglets were exposed to 10% O(2) (hypoxia; n=8) or group B streptococci infusion (GBS; n=5) to produce pulmonary hypertension and were then exposed to 20 ppm NO. RESULTS: NO decreased (>50%) pulmonary artery pressure and vascular resistance in both hypoxia- and GBS-induced pulmonary hypertension, with minimal effects on systemic arterial pressure and cardiac output. NO administration could be performed without detectable environmental leakage. CONCLUSION: Hood NO administration is feasible and shows hemodynamic efficacy in neonatal piglets with pulmonary hypertension.