Bumetanide Suppression of Angiogenesis in a Rat Model of Oxygen-Induced Retinopathy.

Aquaporins (AQPs) are involved in hypoxia-induced angiogenesis and retinal damage. Bumetanide is a diuretic agent, Na+/K+/Cl- cotransporter (NKCC1), and AQP 1-4 inhibitor. We tested the hypothesis that early postnatal treatment with bumetanide suppresses biomarkers of angiogenesis and decreases severe retinopathy oxygen-induced retinopathy (OIR). Neonatal rats were exposed at birth (P0) to either (1) room air (RA); (2) hyperoxia (50% O2); or (3) intermittent hypoxia (IH) consisting of 50% O2 with brief, clustered episodes of 12% O2 from P0 to postnatal day 14 (P14), during which they were treated intraperitoneally (IP) with bumetanide (0.1 mg/kg/day) or an equivalent volume of saline, on P0-P2. Pups were examined at P14 or allowed to recover in RA from P14-P21. Retinal angiogenesis, morphometry, pathology, AQPs, and angiogenesis biomarkers were determined at P14 and P21. Bumetanide reduced vascular abnormalities associated with severe OIR. This was associated with reductions in AQP-4 and VEGF. Bumetanide suppressed sVEGFR-1 in the serum and vitreous fluid, but levels were increased in the ocular tissues during recovery. Similar responses were noted for IGF-I. In this model, early systemic bumetanide administration reduces severe OIR, the benefits of which appear to be mediated via suppression of AQP-4 and VEGF. Further studies are needed to determine whether bumetanide at the right doses may be considered a potential pharmacologic agent to treat retinal neovascularization.

Human retinal endothelial cells and astrocytes cultured on 3-D scaffolds for ocular drug discovery and development.

Topical ocular ketorolac improves the outcomes of severe retinopathy of prematurity and when administered with systemic caffeine, decreases the severity of oxygen-induced retinopathy. We tested the hypothesis that co-cultures of human retinal endothelial cells (HRECs) and human retinal astrocytes (HRAs) on 3-dimensional (3-D) hydrogel scaffolds is a more representative biomimetic paradigm of the blood-retinal-barrier (BRB) than 2-D cultures, and should be utilized for preclinical drug discovery and development. Mono- and co-cultures of HRECs and HRAs were treated with standard doses of ketorolac, ibuprofen, and/or caffeine, and exposed to hyperoxia, intermittent hypoxia (IH), or normoxia on 2-D surfaces or 3-D biodegradable hydrogel scaffolds (AlgiMatrix or Geltrex). Media and cells were collected at 72h post treatment for arachidonic acid metabolites. Cells cultured on 3-D scaffolds exhibited less oxidative stress and variability in drug responses. HRAs enhanced the responses of HRECs to drugs and changes in oxygen environment. PGE2 and PGI2 were the predominant prostanoids produced in response to IH, reflecting COX-2 immunoreactivity. We conclude that HRECs and HRAs co-cultured on 3-D scaffolds may recapitulate drug responses of the dynamic BRB and therefore should be implemented for preclinical ocular drug discovery and development.

Intermittent hypoxia alters dose dependent caffeine effects on renal prostanoids and receptors in neonatal rats.

Caffeine, one of the most commonly prescribed drugs in preterm neonates, is given in standard or suprapharmacologic doses. Although known as a diuretic, its effects in the neonatal kidneys are not well studied. We tested the hypothesis that neonatal intermittent hypoxia (IH) and high caffeine doses (HCD) alter renal regulators of vasomotor tone and water balance. Newborn rats were randomized to room air, hyperoxia, or IH and treated with standard or high caffeine doses; or placebo saline. Renal prostanoids; histopathology; and cyclooxygenase (COX), prostanoid receptor, and aquaporin (AQP) immunoreactivity were determined. HCD in IH caused severe pathological changes in the glomeruli and proximal tubules, consistent with acute kidney injury. This was associated with reductions in anthropometric growth, PGI2, and IP, DP, and AQP-4 immunoreactivity, well as a robust increase in COX-2, suggesting that the use of HCD should be avoided in preterm infants who experience frequent IH episodes.

Oxygen-Induced Retinopathy from Recurrent Intermittent Hypoxia Is Not Dependent on Resolution with Room Air or Oxygen, in Neonatal Rats.

Preterm infants often experience intermittent hypoxia (IH) with resolution in room air (RA) or hyperoxia (Hx) between events. Hypoxia is a major inducer of vascular endothelial growth factor, which plays a key role in normal and aberrant retinal angiogenesis. This study tested the hypothesis that neonatal IH which resolved with RA is less injurious to the immature retina than IH resolved by Hx between events. Newborn rats were exposed to: (1) Hx (50% O2) with brief hypoxia (12% O2); (2) RA with 12% O2; (3) Hx with RA; (4) Hx only; or (5) RA only, from P0 to P14. Pups were examined at P14 or placed in RA until P21. Retinal vascular and astrocyte integrity; retinal layer thickness; ocular and systemic biomarkers of angiogenesis; and somatic growth were determined at P14 and P21. All IH paradigms resulted in significant retinal vascular defects, disturbances in retinal astrocyte template, retinal thickening, and photoreceptor damage concurrent with elevations in angiogenesis biomarkers. These data suggest that the susceptibility of the immature retina to changes in oxygen render no differences in the outcomes between RA or O2 resolution. Interventions and initiatives to curtail O2 variations should remain a high priority to prevent severe retinopathy.

Intravitreal bevacizumab alters type IV collagenases and exacerbates arrested alveologenesis in the neonatal rat lungs.

Purpose/Aim: Intravitreal bevacizumab (Avastin) is an irreversible vascular endothelial growth factor (VEGF) inhibitor used off-label to treat severe retinopathy of prematurity in extremely low gestational age neonates. VEGF and matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) participate in lung maturation. We tested the hypothesis that intravitreal bevacizumab enters the systemic circulation and has long-lasting effects on lung MMPs. MATERIALS AND METHODS: Neonatal rats were exposed to: (1) hyperoxia (50% O2); (2) intermittent hypoxia (IH) (50% O2 with brief episodes of 12% O2); or (3) room air (RA) from birth (P0) to P14. At P14, the time of eye opening in rats, a single dose of Avastin (0.125 mg) was injected into the vitreous cavity of the left eye. A control group received equivalent volume saline. At P23 and P45, lung MMP-2 and MMP-9, and TIMP-1, and TIMP-2 were assessed in the lungs. RESULTS: At P23, Avastin increased MMP-2, MMP-9, and TIMP-1 levels in the hyperoxia group but decreased TIMP-1 levels in the IH group. The ratios of MMP-2/TIMP-1 and MMP-9/TIMP-1 were significantly elevated at P23 in the IH group treated with Avastin. At P45, the levels of MMP-2 and MMP-9 remained elevated in the hyperoxia and IH groups treated with Avastin, while a rebound increase in TIMP-1 levels was noted in the IH group. CONCLUSIONS: Avastin treatment in IH has lasting alterations in the balance between MMPs and their tissue inhibitors. These changes may lead to impaired alveologenesis and tissue damage consistent with bronchopulmonary dysplasia/chronic lung disease.

Pharmacologic synergism of ocular ketorolac and systemic caffeine citrate in rat oxygen-induced retinopathy.

BACKGROUND: Caffeine or ketorolac decrease the risk of retinopathy of prematurity and may act synergistically to improve beneficial effect. Combination of caffeine (Caff) and ketorolac (Keto) to prevent oxygen-induced retinopathy was studied. METHODS: Newborn rats exposed to room air (RA) or intermittent hypoxia (IH) consisting of 12% O2 during hyperoxia (50% O2) from birth (P0) had single daily IP injections of Caff from P0-P13 or saline; and/or ocular Keto (Acuvail, 0.45% ophthalmic solution) administered subcutaneously over the eyes from P5-P7. Pups were studied at P14 or placed in RA for recovery from IH (IHR) until P21. Eyes were examined for neovascularization, histopathology, growth factors, and VEGF-signaling genes. RESULTS: Severe retinal damage noted during IHR in the untreated groups evidenced by hemorrhage, neovascularization, and oxygen-induced retinopathy (OIR) pathologies were prevented with Keto/Caff treatment. Keto and/or Caff treatment in IH also promoted retinal neural development evidenced by eye opening (92%, P < 0.001 vs. 31% in the placebo-treated IH group). No corneal pathologies were noted with Keto. CONCLUSION: Caff or Keto given individually reduced retinal neovascularization, but the two drugs given together prevented severe OIR.

Response of vascular endothelial growth factor and angiogenesis-related genes to stepwise increases in inspired oxygen in neonatal rat lungs.

BACKGROUND: Bronchopulmonary dysplasia is an inflammatory lung disease that afflicts preterm infants requiring supplemental oxygen and is associated with impaired pulmonary angiogenesis. We tested the hypothesis that there is a critical threshold of inspired O2 (FiO2) that alters pulmonary angiogenesis. METHODS: Within 2-6 h of birth, rat pups were exposed to 10%, 21%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% FiO2 for 2 h. Mixed arterial-venous blood gases, serum and pulmonary levels of vascular endothelial growth factor (VEGF) and soluble VEGF receptor-1, and pulmonary angiogenesis gene profiles were determined. RESULTS: PO2 increased with hyperoxia from 35.6 ± 5.0 (range: 31.5-39.8) at 10% O2 to 108.5 ± 25.0 (range: 82.2-134.8) at 100% O2. PO2 at 21% O2 was 42.4 ± 7.3 (range: 36.8-48.1). Lung VEGF levels declined at 40%-100%. The critical PO2 associated with decreased lung VEGF was 66 mm Hg, achieved with a FiO2 of 0.4. PO2 was inversely correlated with VEGF levels in the lungs (R = -0.377; P < 0.008). Antiangiogenesis genes were robustly upregulated at 70%, predominantly in males. Data are reported as mean ± SD. CONCLUSIONS: A critical threshold of FiO2 affecting angiogenesis exists in immature lungs. Exposure of preterm lungs to >40% inspired O2, even for 2 h, may result in abnormal expression of biomarkers regulating lung angiogenesis.

Pharmacologic interventions for the prevention and treatment of retinopathy of prematurity.

Retinopathy of Prematurity (ROP) is a preventable neovascular retinal disease with a lifetime impact on vision and ocular morbidities. Retinal vessel immaturity and oxygen therapy, influenced or modulated by several risk factors including oxidative stress, intermittent hypoxia and desaturations, inflammation, infection, malnutrition, retinal growth factor deficiencies or excesses, and others are determinant factors of pathologic retinal angiogenesis and ROP. These factors are pharmacologic targets for prevention and/or rescue therapy. These drugs, include intravitreal anti-vascular endothelial growth factor drugs, erythropoietin, ocular propranolol, caffeine, antioxidants, insulin-like growth factor-I, and omega 3 poly-unsaturated fatty acids, and are promising therapies to prevent ROP, but require further studies. Topical ocular non-steroidal anti-inflammatory drugs (NSAIDs) target inflammatory cascade but the best, safest, and most effective ocular NSAID and formulation remain to be developed. Timing of drug intervention appears critical. Moreover, the complex interactions of the various pathophysiologic mechanisms resulting in aberrant angiogenesis thence ROP strongly suggest that drug combinations and synergisms may be required for effective prevention of ROP and a lifetime of blindness.

Impact of Chronic Neonatal Intermittent Hypoxia on Severity of Retinal Damage in a Rat Model of Oxygen-Induced Retinopathy.

Neonatal intermittent hypoxia (IH) followed by re-oxygenation in normoxia or supplemental oxygen (IHR) increases the risk for severe retinopathy of prematurity (ROP). The exact timing for the onset of retinal damage which may guide strategic interventions during retinal development, is unknown. We tested the hypothesis that chronic exposure of the immature retina to neonatal IH induces early manifestations of retinal damage that can be utilized as key time points for strategic pharmacologic intervention. Newborn rats were exposed to IH within 2 hours of birth (P0) until P14, or allowed to recover in room air (RA) from P14 to P21 (IHR). Retinal integrity and angiogenesis biomarkers were progressively assessed before (P0), during IH, and post IH (recovery in RA), or IHR, and compared to normoxic age-matched controls. Retinal damage occurred as early as day 3 of neonatal IH, consistent with vascular abnormalities and disturbances in the astrocytic template. These abnormalities worsened during IHR. Pharmacologic and non-pharmacologic interventions to identify, prevent, or minimize neonatal IH should be implemented shortly after birth in high risk preterm newborns. This strategy may lead to a reduction in the outcome of severe ROP requiring later invasive treatments.

Intermittent hypoxia suppression of growth hormone and insulin-like growth factor-I in the neonatal rat liver.

OBJECTIVES: Extremely low gestational age neonates with chronic lung disease requiring oxygen therapy frequently experience fluctuations in arterial oxygen saturation or intermittent hypoxia (IH). These infants are at risk for multi-organ developmental delay, reduced growth, and short stature. The growth hormone (GH)/insulin-like growth factor-I (IGF-1) system, an important hormonal regulator of lipid and carbohydrate metabolism, promotes neonatal growth and development. We tested the hypothesis that increasing episodes of IH delay neonatal growth by influencing the GH/IGF-I axis. DESIGN: Newborn rats were exposed to 2, 4, 6, 8, 10, or 12 hypoxic episodes (12% O2) during hyperoxia (50% O2) from P0-P7, P0-P14 (IH), or allowed to recover from P7-P21 or P14-P21 (IHR) in room air (RA). RA littermates at P7, P14, and P21 served as RA controls; and groups exposed to hyperoxia only (50% O2) served as zero IH controls. Histopathology of the liver; hepatic levels of GH, GHBP, IGF-I, IGFBP-3, and leptin; and immunoreactivities of GH, GHR, IGF-I and IGF-IR were determined. RESULTS: Pathological findings of the liver, including cellular swelling, steatosis, necrosis and focal sinusoid congestion were seen in IH, and were particularly severe in the P7 animals. Hepatic GH levels were significantly suppressed in the IH groups exposed to 6-12 hypoxic episodes per day and were not normalized during IHR. Deficits in the GH levels were associated with reduced body length and increase body weight during IHR suggesting increased adiposity and catchup fat. Catchup fat was also associated with elevations in GHBP, IGF-I, leptin. CONCLUSIONS: IH significantly impairs hepatic GH/IGF-1 signaling during the first few weeks of life, which is likely responsible for hepatic GH resistance, increased body fat, and hepatic steatosis. These hormonal perturbations may contribute to long-term organ and body growth impairment, and metabolic dysfunction in preterm infants experiencing frequent IH and/or apneic episodes.

Comparative Effects of Coenzyme Q10 or n-3 Polyunsaturated Fatty Acid Supplementation on Retinal Angiogenesis in a Rat Model of Oxygen-Induced Retinopathy.

Neonatal intermittent hypoxia (IH) or apnea afflicts 70% to 90% of all preterm infants <28 weeks gestation, and is associated with severe retinopathy of prematurity (ROP). We tested the hypotheses that coenzyme Q10 (CoQ10) or omega-3 polyunsaturated fatty acids (n-3 PUFAs) supplementation during neonatal IH reduces the severity of oxygen-induced retinopathy (OIR). Newborn rats were exposed to two IH paradigms: (1) 50% O2 with brief hypoxia (12% O2); or (2) 21% O2 with brief hypoxia, until postnatal day 14 (P14), during which they received daily oral CoQ10 in olive oil, n-3 PUFAs in fish oil, or olive oil only and compared to room air (RA) treated groups. Pups were examined at P14, or placed in RA until P21. Retinal angiogenesis, histopathology, and morphometry were determined. Both IH paradigms produced severe OIR, but these were worsened with 50/12% O2 IH. CoQ10 and n-3 PUFAs reduced the severity of OIR, as well as ocular growth factors in both IH paradigms, but CoQ10 was more effective in 50/12% O2 IH. Supplementation with either CoQ10 or n-3 PUFAs targeting IH-induced retinal injury is individually effective for ameliorating specific characteristics consistent with ROP. Given the complexity of ROP, further studies are needed to determine whether combined CoQ10 and n-3 PUFAs supplementation would optimize their efficacy and result in a better outcome.

Non-steroidal Anti-inflammatory Drugs in Newborns and Infants.

Nonsteroidal antiinflammatory drugs (NSAIDs) and acetaminophen are used in young infants and newborns for pain and fever control, patent ductus closure, prevention of intraventricular hemorrhage, and potentially for prevention of retinopathy of prematurity. These drugs inhibit cyclooxygenase 1 (COX-1), COX-2, and peroxidases, thus, blocking prostaglandin (PG) synthesis. PGs are eicosanoids that regulate several physiologic, pathologic, and cellular processes, including vasomotor tone, platelet aggregation, sensitization of neurons to pain, and many molecular events critical to physiologic homeostasis. NSAIDs inhibit caspases and cell death. Increasing knowledge of these molecular entities may allow targeted drug development to prevent or minimize neonatal morbidities.

MnTBAP or Catalase Is More Protective against Oxidative Stress in Human Retinal Endothelial Cells Exposed to Intermittent Hypoxia than Their Co-Administration (EUK-134).

Retinopathy of prematurity is a blinding disease that affects extremely low gestational age neonates. Its etiology is due to extrauterinehyperoxia in an immature antioxidant system culminating as oxidative stress on the retina. Our aim is to elucidate the role of pharmacological antioxidants in modulating the biochemical and molecular response of human retinal microvascular endothelial cells (HRECs) exposed to oxidative stress. HRECs were treated with MnTBAP [a superoxide dismutase (SOD) mimetic], catalase, EUK-134 (SOD + catalase), or saline prior to exposure to normoxia (Nx), hyperoxia (Hx), or intermittent hypoxia (IH). Media levels of SOD, catalase, glutathione peroxidase (GPx), 8-isoPGF2α, and H2O2; cellular SOD and catalase; cellular function (migration and tube formation); and antioxidant gene expression were assessed. Pharmacological antioxidants had delayed suppressive effect on 8-isoPGF2α. MnTBAP and catalase were more effective for H2O2 scavenging in the media than co-administration in the form of EUK-134. A delayed response was noted in SOD and catalase media activity in MnTBAP- and catalase-treated cells, respectively in 50% and IH. MnTBAP had progressively increased media GPx in all oxygen conditions. Antioxidants resulted in normal, but more abundant tubulogenesis in IH and Hx. The distinct temporal response to oxidative stress reflected the respective antioxidant's potency and catalytic properties. The cell permeability of the antioxidants limited the ability to scavenge intracellular free radicals. The results support that MnTBAP or catalase may be more effective for the prevention of oxidative stress in oxygen-induced retinopathy.

Chronic Intermittent Hypoxia Causes Lipid Peroxidation and Altered Phase 1 Drug Metabolizing Enzymes in the Neonatal Rat Liver.

Critically ill preterm neonates requiring oxygen therapy often experience frequent apneas with intermittent hypoxia (IH). IH-induced oxidative stress causes lipid peroxidation, which targets the liver and contributes to toxic drug reactions. We tested the hypothesis that incremental IH episodes induce oxidative damage in the neonatal liver and alter the expression of genes that regulate drug metabolism. Newborn rats were exposed to increasing IH episodes (12% O2) during hyperoxia (50% O2), or placed in room air (RA) until postnatal day 21 (P21) for recovery from IH (IHR). RA littermates served as controls, and pups exposed to 50% O2 served as hyperoxia controls. Hepatic histopathology, biomarkers of oxidative stress and oxidative DNA damage, antioxidants, and expression of genes that regulate drug metabolism were assessed. Oxidative stress and DNA damage, evidenced by 8-isoprostaglandin F2α (8-isoPGF2α) and 8-hydroxy-2'-deoxyguanosine (8-OH-dG), respectively, increased as a function of IH episodes, and was associated with decreased superoxide dismutase (SOD) and increased catalase activities. Pathological changes including cellular swelling, steatosis, necrosis, and focal sinusoid congestion were seen in IH, but not in IHR. Similarly, IH was associated with upregulation of several genes involved in DNA repair, which were downregulated during IHR. Of the genes involved in drug metabolism, aldehyde dehydrogenases (involved in lipid peroxidation) and cytochrome P450 (CYP) genes of the 2C family (involved in oxidative stress) were robustly upregulated both in IH and in IHR. Hepatic oxidative stress and lipid peroxidation occurring in response to chronic IH have implications for preterm infants, and may explain, in part, the pharmacokinetic variations and drug toxicities in this vulnerable population.

Co-Enzyme Q10 and n-3 Polyunsaturated Fatty Acid Supplementation Reverse Intermittent Hypoxia-Induced Growth Restriction and Improved Antioxidant Profiles in Neonatal Rats.

Neonatal intermittent hypoxia (IH) increases the risk for many morbidities in extremely low birth weight/gestational age (ELBW/ELGA) neonates with compromised antioxidant systems and poor growth. We hypothesized that supplementation with coenzyme Q10 (CoQ10, ubiquinol) or n-3 polyunsaturated fatty acids (PUFAs) during neonatal IH improves antioxidant profiles and somatic growth in neonatal rats. Newborn rats were exposed to two IH paradigms at birth (P0): (1) 50% O2 with brief hypoxic episodes (12% O2); or (2) room air (RA) with brief hypoxia, until P14 during which they received daily oral CoQ10 in olive oil, n-3 PUFAs in fish oil, or olive oil only from P0 to P14. Pups were studied at P14 or placed in RA until P21 for recovery from IH (IHR). Body weight and length; organ weights; and serum antioxidants and growth factors were determined at P14 and P21. Neonatal IH resulted in sustained reductions in somatic growth, an effect that was reversed with n-3 PUFAs. Improved growth was associated with higher serum growth factors. CoQ10 decreased superoxide dismutase (SOD) and glutathione, but increased catalase, suggesting reduced oxidative stress. Further studies are needed to determine the synergistic effects of CoQ10 and n-3 PUFA co-administration for the prevention of IH-induced oxidative stress and postnatal growth deficits.

Neonatal Intermittent Hypoxia, Reactive Oxygen Species, and Oxygen-Induced Retinopathy.

Most of the major morbidities in the preterm newborn are caused by or are associated with oxygen-induced injuries and are aptly called "oxygen radical diseases in neonatology or ORDIN". These include bronchopulmonary dysplasia, retinopathy of prematurity, periventricular leukomalacia, intraventricular hemorrhage, necrotizing enterocolitis and others. Relative hyperoxia immediately after birth, immature antioxidant systems, biomolecular events favoring oxidative stress such as iron availability and the role of hydrogen peroxide as a key molecular mediator of these events are reviewed. Potential therapeutic strategies such as caffeine, antioxidants, non-steroidal anti-inflammatory drugs, and others targeted to these critical sites may help prevent oxidative radical diseases in the newborn resulting in improved neonatal outcomes.

Pharmacologic interventions for the prevention and treatment of retinopathy of prematurity.

Retinopathy of prematurity (ROP), a significant morbidity in prematurely born infants, is the most common cause of visual impairment and blindness in children and persists till adulthood. Strict control of oxygen therapy and prevention of intermittent hypoxia are the keys in the prevention of ROP, but pharmacologic interventions have decreased risk of ROP. Various drug classes such as methylxanthines (caffeine), VEGF inhibitors, antioxidants, and others have decreased ROP occurrence. The timing of pharmacologic intervention remains unsettled, but early prevention rather than controlling disease progression may be preferred. These drugs act through different mechanisms, and synergistic approaches should be considered to maximize efficacy and safety.

Exogenous Superoxide Dismutase Mimetic Without Scavenging H2O2 Causes Photoreceptor Damage in a Rat Model for Oxygen-Induced Retinopathy.

PURPOSE: Frequent, brief intermittent episodes of hypoxia (IH) during hyperoxia increase reactive oxygen species in the immature retina with compromised antioxidant systems, thus leading to oxygen-induced retinopathy (OIR). We examined the hypothesis that early exposure to a mimetic of superoxide dismutase (SOD), the first line of defense against oxidative stress, will decrease IH-induced reactive oxygen species (ROS) and prevent severe OIR in our rat model. METHODS: To test this hypothesis, newborn rats (P0) were exposed to IH consisting of alternating cycles of 50% O2 with brief hypoxia (12% O2) until P14 during which they were treated with a single daily intraperitoneal (IP) dose of MnTBAP (a SOD mimetic) at 1.0, 5.0, or 10.0 mg/kg on P0, P1, and P2. A saline-treated group served as vehicle controls. Groups were analyzed following IH at P14 or allowed to recover in room air (RA) until P21. Control littermates were raised in RA with all conditions identical except for inspired O2. Ocular assessment of OIR severity, oxidative stress, angiogenesis, antioxidant activity, and oxidative phosphorylation (OXPHOS) were conducted at P14 and P21. RESULTS: Collectively, the data show increased oxidative stress and angiogenesis with MnTBAP, which was associated with photoreceptor damage, retinal characteristics consistent with severe OIR, and changes in genes regulating OXPHOS. CONCLUSIONS: In the setting of IH, the use of exogenous SOD mimetics must be combined with H2O2 scavengers in order to prevent photoreceptor damage and severe OIR.

Benefits of pre-, pro- and Syn-biotics for lung angiogenesis in malnutritional rats exposed to intermittent hypoxia.

Extremely low birth weight and reduced caloric intake have significant adverse effects on lung development and are risk factors for bronchopulmonary dysplasia. Vascular endothelial growth factor (VEGF) is highly involved in lung microvascular development, and may be affected by nutritional status. To test the hypothesis that suboptimal nutrition decreases VEGF signaling in formula-fed neonatal rats, and to determine whether supplementation with probiotics, prebiotics, or synbiotics ameliorate the effects, rat pups at birth (P0) were placed in room air (RA) or intermittent hypoxia (12%) during hyperoxia (50% O2) from birth to P3. The pups were either maternally-fed; or formula-fed with or without supplementation. Formula-fed pups were separated from their mothers at birth and hand-gavaged every 3 hours. Lung VEGF signaling was determined on P3. In RA, all formula-fed groups were significantly growth suppressed with decreased lung weights. Hyperoxia had a less remarkable effect on body weight; and mean lung weight was lower only in the unsupplemented formula-fed group. Lung VEGF was decreased in all formula-fed RA and hyperoxia groups, except the probiotics group. In RA, sVEGFR-1 levels were elevated in all formula-fed groups except the synbiotics group. However in hyperoxia, sVEGFR-1 levels were higher in the unsupplemented formula group. All genes involved in angiogenesis were downregulated in the formula-fed groups compared to maternally-fed. Formula feeding results in significant malnutrition associated with decreased lung size and lung VEGF levels in neonatal rat pups. Probiotic supplementation prevented the adverse effects of combined hyperoxia and suboptimal nutrition on lung VEGF suggesting preservation of angiogenesis.

Hydrogen peroxide accumulation in the choroid during intermittent hypoxia increases risk of severe oxygen-induced retinopathy in neonatal rats.

PURPOSE: Extremely low gestational age neonates (ELGANs) requiring oxygen therapy often experience frequent episodes of intermittent hypoxia (IH) and are at high risk for severe retinopathy of prematurity (ROP). Using an established model for oxygen-induced retinopathy (OIR), we examined the hypothesis that there is a critical number of daily brief IH episodes which will result in irreversible retinal oxidative damage. METHODS: Newborn rats were exposed to increasing daily clustered IH episodes (12% O2 with 50% O2) from postnatal day (P) 0 to P7 or P0 to P14, or placed in room air (RA) until P21 following 7- or 14-day IH. RA littermates at P7, P14, and P21 served as controls. A group exposed to constant 50% O2 (CH) served as a second control. Blood gases, eye opening at P14, retinal, and choroidal oxidative stress and lipid peroxidation (8-isoPGF(2α)), oxidants (H2O2) and antioxidants (catalase and SOD), retinal pathology (adenosine diphosphatase (ADPase)-stained retinal flatmounts), and mitochondria-related genes were assessed. RESULTS: pO2 levels were higher with increasing IH episodes and remained elevated during the reoxygenation period. High SO2 levels were associated with most severe OIR. Levels of all measured biomarkers peaked with six IH episodes and decreased with 8 to 12 episodes. H2O2 accumulated in the choroid during the reoxygenation period with irreversible retinal damage. CONCLUSIONS: Our data suggest that six is the maximum number of IH episodes that the retina can sustain. Accumulation of H2O2 in the choroid may result in high levels being delivered to the entire retina, ultimately resulting in irreversible retinal oxidative damage.