Comparison of Glutathione Nanoparticles, CoEnzyme Q10, and Fish Oil for Prevention of Oxygen-Induced Retinopathy in Neonatal Rats.

Notch ligands and receptors are important for cell specification and angiogenesis, but their role in oxygen-induced retinopathy (OIR) is not well studied. Delta-like ligand (DLL)-4/Notch inhibits angiogenesis, while Jagged-1/Notch promotes angiogenesis. We tested the hypothesis that early supplementation with antioxidants and/or fish oil curtails severe OIR by inducing DLL-4/Notch and reducing Jagged-1/Notch. Newborn rats were exposed to brief intermittent hypoxia (IH) during hyperoxia, during which they received daily oral supplements of (1) fish oil, (2) coenzyme Q10 (CoQ10) in olive oil (OO), (3) glutathione nanoparticles (nGSH), (4) fish oil + CoQ10, or (5) OO (controls) from birth (P0) to P14. At P14, the pups were placed in room air (RA) until P21, with no further treatment. Oxidative stress, apoptosis, ocular histopathology, and Notch signaling were assessed. Neonatal IH resulted in severe retinal damage consistent with retinopathy of prematurity (ROP). Retinal damage was associated with induced oxidative stress and Jagged-1/Notch signaling, as well as reduced DLL-4/Notch signaling. All treatments reversed these outcomes, but nGSH produced the most beneficial outcomes. Severe OIR promoted the induction of Jagged-1/Notch and curtailed DLL-4/Notch, which was an effect that could be reversed with nGSH supplementation. These findings may indicate a potential alternate pathway for ROP treatment and/or prevention.

Early versus late caffeine and/or non-steroidal anti-inflammatory drugs (NSAIDS) for prevention of intermittent hypoxia-induced neuroinflammation in the neonatal rat.

Preterm infants often experience frequent intermittent hypoxia (IH) episodes which are associated with neuroinflammation. We tested the hypotheses that early caffeine and/or non-steroidal inflammatory drugs (NSAIDs) confer superior therapeutic benefits for protection against IH-induced neuroinflammation than late treatment. Newborn rats were exposed to IH or hyperoxia (50% O2) from birth (P0) to P14. For early treatment, the pups were administered: 1) daily caffeine (Caff) citrate (Cafcit, 20 mg/kg IP loading on P0, followed by 5 mg/kg from P1-P14); 2) ketorolac (Keto) topical ocular solution in both eyes from P0 to P14; 3) ibuprofen (Ibu, Neoprofen, 10 mg/kg loading dose on P0 followed by 5 mg/kg/day on P1 and P2); 4) Caff+Keto co-treatment; 5) Caff+Ibu co-treatment; or 6) equivalent volume saline (Sal). On P14, animals were placed in room air (RA) with no further treatment until P21. For late treatment, pups were exposed from P0 to P14, then placed in RA during which they received similar treatments from P15-P21 (Sal, Caff, and/or Keto), or P15-P17 (Ibu). RA controls were similarly treated. At P21, whole brains were assessed for histopathology, apoptosis, myelination, and biomarkers of inflammation. IH caused significant brain injury and hemorrhage, inflammation, reduced myelination, and apoptosis. Early treatment with Caff alone or in combination with NSAIDs conferred better neuroprotection against IH-induced damage than late treatment. Early postnatal treatment during a critical time of brain development, may be preferable for the prevention of IH-induced brain injury in preterm infants.

Factors Influencing Neonatal Gut Microbiome and Health with a Focus on Necrotizing Enterocolitis.

Maturational changes in the gut start in utero and rapidly progress after birth, with some functions becoming fully developed several months or years post birth including the acquisition of a full gut microbiome, which is made up of trillions of bacteria of thousands of species. Many factors influence the normal development of the neonatal and infantile microbiome, resulting in dysbiosis, which is associated with various interventions used for neonatal morbidities and survival. Extremely low gestational age neonates (<28 weeks' gestation) frequently experience recurring arterial oxygen desaturations, or apneas, during the first few weeks of life. Apnea, or the cessation of breathing lasting 15-20 s or more, occurs due to immature respiratory control and is commonly associated with intermittent hypoxia (IH). Chronic IH induces oxygen radical diseases of the neonate, including necrotizing enterocolitis (NEC), the most common and devastating gastrointestinal disease in preterm infants. NEC is associated with an immature intestinal structure and function and involves dysbiosis of the gut microbiome, inflammation, and necrosis of the intestinal mucosal layer. This review describes the factors that influence the neonatal gut microbiome and dysbiosis, which predispose preterm infants to NEC. Current and future management and therapies, including the avoidance of dysbiosis, the use of a human milk diet, probiotics, prebiotics, synbiotics, restricted antibiotics, and fecal transplantation, for the prevention of NEC and the promotion of a healthy gut microbiome are also reviewed. Interventions directed at boosting endogenous and/or exogenous antioxidant supplementation may not only help with prevention, but may also lessen the severity or shorten the course of the disease.

Comparison of hyperoxia or normoxia resolution of intermittent hypoxia and intermittent hyperoxia episodes on liver histopathology, IGF-1, IGFBP-3, and GHBP in neonatal rats.

OBJECTIVE: Extremely low gestational age neonates requiring oxygen therapy for chronic lung disease experience repeated fluctuations in arterial oxygen saturation, or intermittent hypoxia (IH), during the first few weeks of life. These events are associated with a high risk for reduced growth, hypertension, and insulin resistance in later life. This study tested the hypothesis that IH, or intermittent hyperoxia have similar negative effects on the liver; somatic growth; and liver insulin-like growth factor (IGF)-I, IGF binding protein (BP)-3, and growth hormone binding protein (GHBP), regardless of resolution in normoxia or hyperoxia between episodes. DESIGN: Newborn rats on the first day of life (P0) were exposed to two IH paradigms: 1) hyperoxia (50% O2) with brief hypoxia (12% O2); or 2) normoxia (21% O2) with hypoxia (12% O2); intermittent hypoxia (50% O2/21% O2); hyperoxia only (50% O2); or room air (RA, 21% O2). Pups were euthanized on P14 or placed in RA until P21. Controls remained in RA from P0-P21. Growth, liver histopathology, apoptosis, IGFI, IGFBP-3, and GHBP were assessed. RESULTS: Pathological findings of the liver hepatocytes, including cellular swelling, steatosis, apoptosis, necrosis and focal sinusoid congestion were seen in the IH and intermittent hyperoxia groups, and were particularly severe in the 21-12% O2 group during exposure (P14) with no significant improvements during recovery/reoxygenation (P21). These effects were associated with induction of HIF1α, and reductions in liver IGFI, IGFBP-3, and GHBP. CONCLUSIONS: Exposure to IH or intermittent hyperoxia during the first few weeks of life regardless of resolution in RA or hyperoxia is detrimental to the immature liver. These findings may suggest that interventions to prevent frequent fluctuations in oxygen saturation during early neonatal life remain a high priority.

Comparison of Bevacizumab and Aflibercept for Suppression of Angiogenesis in Human Retinal Microvascular Endothelial Cells.

Bevacizumab (Avastin) is a vascular endothelial growth factor (VEGF) inhibitor that is widely used for aggressive posterior retinopathy of prematurity (APROP). Its use is associated with multiple adverse effects. Aflibercept (Eylea) is a VEGFR-1 analogue that is approved for ocular use, but its efficacy for APROP is less studied. We tested the hypothesis that Eylea is as effective as Avastin for suppression of intermittent hypoxia (IH)-induced angiogenesis. Human retinal microvascular endothelial cells (HRECs) were treated with Avastin and low- or high-dose Eylea and exposed to normoxia, hyperoxia (50% O2), or neonatal IH for 24, 48, or 72 h. Cells were assessed for migration and tube formation capacities, as well as biomarkers of angiogenesis and oxidative stress. Both doses of Eylea suppressed migration and tube formation in all oxygen environments, although the effect was not as robust as Avastin. Furthermore, the lower dose of Eylea appeared to be more effective than the higher dose. Eylea induced soluble VEGFR-1 (sVEGFR-1) coincident with high IGF-I levels and decreased Notch/Jagged-1, demonstrating a functional association. Given the role of VEGFR-1 and Notch as guidance cues for vascular sprouting, these data suggest that Eylea may promote normal vascular patterning in a dose-dependent manner.

Biomarkers of lung alveolarization and microvascular maturation in response to intermittent hypoxia and/or early antioxidant/fish oil supplementation in neonatal rats.

OBJECTIVE: Extremely preterm infants experience frequent intermittent hypoxia (IH) episodes during oxygen therapy which causes significant damage to the lungs and curtails important signaling pathways that regulate normal lung alveolarization and microvascular maturation. We tested the hypothesis that early supplementation with fish oil and/or antioxidants in rats exposed to neonatal IH improves expression of lung biomarkers of alveolarization and microvascular maturation, and reduces IH-induced lung injury. STUDY DESIGN/METHODS: From birth (P0) to P14, rat pups were exposed to room air (RA) or neonatal IH during which they received daily oral supplementation with either: (1) olive oil (OO) (control); (2) Coenzyme Q10 (CoQ10) in OO; (3) fish oil; (4) glutathione nanoparticles (nGSH); or (5) fish oil +CoQ10. At P14 pups were placed in RA until P21 with no further treatment. RA controls were similarly treated. Lung growth and alveolarization, histopathology, apoptosis, oxidative stress and biomarkers of alveolarization and microvascular maturation were determined. RESULTS: Neonatal IH was associated with reduced lung weights and severe histopathological outcomes. These effects were curtailed with fish oil and nGSH. nGSH was also protective against apoptosis, while CoQ10 prevented IH-induced ROS production. Of all treatments, nGSH and CoQ10 + fish oil-induced vascular endothelial growth factor165 and CD31 (Platelet endothelial cell adhesion molecule-1), which are associated with angiogenesis. CoQ10 + fish oil improved alveolarization in RA and IH despite evidence of hemorrhage. CONCLUSIONS: The benefits of nGSH and CoQ10 + fish oil suggest an antioxidant effect which may be required to curtail IH-induced lung injury. Further clinical assessment of the effectiveness of nGSH is warranted.

Biomarkers of growth and carbohydrate metabolism in neonatal rats supplemented with fish oil and/or antioxidants during intermittent hypoxia.

OBJECTIVE: Extremely low gestational age neonates (ELGANs) experience frequent intermittent hypoxia (IH) episodes during therapeutic oxygen. ELGANs exhibit poor postnatal growth requiring lipid supplementation. Lipids are targets of reactive oxygen species resulting in lipid peroxidation and cell death, particularly in preterm infants with compromised antioxidant systems. We tested the hypothesis that early supplementation with lipids and/or antioxidants promotes growth and influences biomarkers of carbohydrate metabolism in neonatal rats exposed to IH. DESIGN: Newborn rats (n = 18/group) were exposed to brief hypoxia (12% O2) during hyperoxia (50% O2), or room air (RA), from birth (P0) to P14 during which they received daily oral supplementation with: 1) fish oil; 2) Coenzyme Q10 (CoQ10) in olive oil; 3) glutathione nanoparticles (nGSH); 4) fish oil+CoQ10; or 5) olive oil. At P21, plasma samples were assessed for glucose, insulin, glucokinase (GCK), glucagon, glucagon-like peptide (GLP)-1, growth hormone (GH), corticosterone, and ghrelin. Liver was assessed for histopathology, apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL stain), and GH, insulin-like growth factor (IGF)-I, GH binding protein (GHBP), and IGF binding protein (IGFBP)-3. RESULTS: Neonatal IH resulted in decreased liver weight and liver/body weight ratios, as well as hepatocyte swelling, steatosis, and apoptosis, which were attenuated with fish oil, nGSH, and combined fish oil+CoQ10. IH also decreased plasma glucose, insulin, GCK, and ghrelin, but increased GLP-1. All treatments improved plasma glucose in IH, but insulin was higher with CoQ10 and nGSH only. Glucagon was increased with CoQ10, fish oil, and CoQ10 + fish oil, while corticosterone was higher with nGSH and CoQ10 + fish oil. IGF-I and IGFBP-3 were significantly higher in the liver with CoQ10 in IH, while deficits in GH were noted with CoQ10 and fish oil in RA and IH. Treatment with nGSH and combined CoQ10 + fish oil reduced IGF-I in RA and IH but increased IGFBP-3. CONCLUSIONS: Neonatal IH impairs liver growth with significant hepatocyte damage. Of all supplements in IH, nGSH and combined fish oil+CoQ10 were most effective for preserving liver growth and carbohydrate metabolism. Data suggest that these supplements may improve poor postnatal organ and body growth; and metabolic dysfunction associated with neonatal IH.

Neonatal intermittent hypoxia, fish oil, and/or antioxidant supplementation on gut microbiota in neonatal rats.

BACKGROUND: Preterm infants frequently experience intermittent hypoxia (IH) episodes, rendering them susceptible to oxidative stress and gut dysbiosis. We tested the hypothesis that early supplementation with antioxidants and/or fish oil promotes gut biodiversity and mitigates IH-induced gut injury. METHODS: Newborn rats were exposed to neonatal IH from birth (P0) to P14 during which they received daily oral supplementation with: (1) coenzyme Q10 (CoQ10) in olive oil, (2) fish oil, (3) glutathione nanoparticles (nGSH), (4) CoQ10 + fish oil, or (5) olive oil (placebo control). Pups were placed in room air (RA) from P14 to P21 with no further treatment. RA controls were similarly treated. Stool samples were assessed for microbiota and terminal ileum for histopathology and morphometry, total antioxidant capacity, lipid peroxidation, and biomarkers of gut injury. RESULTS: Neonatal IH induced histopathologic changes consistent with necrotizing enterocolitis, which were associated with increased lipid peroxidation, toll-like receptor, transforming growth factor, and nuclear factor kappa B. Combination of CoQ10 + fish oil and nGSH were most effective for preserving gut integrity, reducing biomarkers of gut injury, and increasing commensal organisms. CONCLUSIONS: Combination of antioxidants and fish oil may confer synergistic benefits to mitigate IH-induced injury in the terminal ileum. IMPACT: Antioxidant and fish oil (PUFA) co-treatment was most beneficial for reducing neonatal IH-induced gut injury. The synergistic effects of antioxidant and fish oil is likely due to prevention of IH-induced ROS attack on lipids, thus preserving and augmenting its therapeutic benefits. Combination treatment was also effective for increasing the abundance of the non-pathogenic Firmicutes phylum, which is associated with a healthy gastrointestinal system of the newborn. Extremely low gestational age neonates who are at high risk for frequent, repetitive neonatal IH and oxidative stress-induced diseases may benefit from this combination therapy.

Dose Response of Bumetanide on Aquaporins and Angiogenesis Biomarkers in Human Retinal Endothelial Cells Exposed to Intermittent Hypoxia.

Aquaporins (AQPs) are important for regulating cellular water, solute transport, and balance. Recently, AQPs have also been recognized as playing a key role in cell migration and angiogenesis. In the retina, hypoxia induces vascular endothelial growth factor (VEGF), a potent angiogenic and vascular permeability factor, resulting in retinal edema, which is facilitated by AQPs. Bumetanide is a diuretic agent and AQP 1-4 blocker. We tested the hypothesis that bumetanide suppression of AQPs ameliorates intermittent hypoxia (IH)-induced angiogenesis and oxidative stress in human microvascular retinal endothelial cells (HMRECs). HMRECs were treated with a low-dose (0.05 µg/mL) or high-dose (0.2 µg/mL) of bumetanide and were exposed to normoxia (Nx), hyperoxia (50% O2), or IH (50% O2 with brief hypoxia 5% O2) for 24, 48, and 72 h. Angiogenesis and oxidative stress biomarkers were determined in the culture media, and the cells were assessed for tube formation capacity and AQP-1 and -4 expression. Both doses of bumetanide significantly decreased oxidative stress and angiogenesis biomarkers. This response was reflected by reductions in tube formation capacity and AQP expression. These findings confirm the role of AQPs in retinal angiogenesis. Therapeutic targeting of AQPs with bumetanide may be advantageous for IH-induced aberrant retinal development.

Renal biomarkers of acute kidney injury in response to increasing intermittent hypoxia episodes in the neonatal rat.

BACKGROUND: We tested the hypotheses that: 1) early exposure to increasing episodes of clinically relevant intermittent hypoxia (IH) is detrimental to the developing kidneys; and 2) there is a critical number of daily IH episodes which will result in irreparable renal damage that may involve angiotensin (Ang) II and endothelin (ET)-1. METHODS: At birth (P0), neonatal rat pups were exposed to brief IH episodes from the first day of life (P0) to P7 or from P0-P14. Pups were either euthanized immediately or placed in room air (RA) until P21. RA littermates served as controls. Kidneys were harvested at P7, P14, and P21 for histopathology; angiotensin converting enzyme (ACE), ACE-2, ET-1, big ET-1, and malondialdehyde (MDA) levels; immunoreactivity of ACE, ACE-2, ET-1, ET-2, ET receptors (ETAR, ETBR), and hypoxia inducible factor (HIF)1α; and apoptosis (TUNEL stain). RESULTS: Histopathology showed increased renal damage with 8-12 IH episodes/day, and was associated with Ang II, ACE, HIF1α, and apoptosis. ACE-2 was not expressed at P7, and minimally increased at P14. However, a robust ACE-2 response was seen during recovery with maximum levels noted in the groups recovering from 8 IH episodes/day. ET-1, big ET-1, ETAR, ETBR, and MDA increased with increasing levels of neonatal IH. CONCLUSIONS: Chronic neonatal IH causes severe damage to the developing kidney with associated elevations in vasoconstrictors, suggesting hypertension, particularly with 8 neonatal IH episodes. ACE-2 is not activated in early postnatal life, and this may contribute to IH-induced vasoconstriction. Therapeutic targeting of ACE and ET-1 may help decrease the risk for kidney injury in the developing neonate to prevent and/or treat neonatal acute kidney injury and/or chronic kidney disease.

Comparison of coenzyme Q10 or fish oil for prevention of intermittent hypoxia-induced oxidative injury in neonatal rat lungs.

BACKGROUND: Neonatal intermittent hypoxia (IH) results in oxidative distress in preterm infants with immature antioxidant systems, contributing to lung injury. Coenzyme Q10 (CoQ10) and fish oil protect against oxidative injury. We tested the hypothesis that CoQ10 is more effective than fish oil for prevention of IH-induced lung injury in neonatal rats. METHODS: Newborn rats were exposed to two clinically relevant IH paradigms at birth (P0): (1) 50% O2 with brief hypoxia (12% O2); or (2) room air (RA) with brief hypoxia (12% O2), until P14 during which they were supplemented with daily oral CoQ10, fish oil, or olive oil from P0 to P14. Pups were studied at P14 or placed in RA until P21 with no further treatment. Lungs were assessed for histopathology and morphometry; biomarkers of oxidative stress and lipid peroxidation; and antioxidants. RESULTS: Of the two neonatal IH paradigms 21%/12% O2 IH resulted in the most severe outcomes, evidenced by histopathology and morphometry. CoQ10 was effective for preserving lung architecture and reduction of IH-induced oxidative stress biomarkers. In contrast, fish oil resulted in significant adverse outcomes including oversimplified alveoli, hemorrhage, reduced secondary crest formation and thickened septae. This was associated with elevated oxidants and antioxidants activities. CONCLUSIONS: Data suggest that higher FiO2 may be needed between IH episodes to curtail the damaging effects of IH, and to provide the lungs with necessary respite. The negative outcomes with fish oil supplementation suggest oxidative stress-induced lipid peroxidation.

Antioxidants and/or fish oil reduce intermittent hypoxia-induced inflammation in the neonatal rat terminal ileum.

Intermittent hypoxia (IH) is associated with the pathogenesis of necrotizing enterocolitis (NEC). We tested the hypothesis that early supplementation with antioxidants and/or fish oil protects the terminal ileum from oxidative injury induced by neonatal IH. Newborn rats were exposed to neonatal IH from birth (P0) until P14 during which they received daily fish oil, coenzyme Q10 (CoQ10), glutathione nanoparticles (nGSH), fish oil + CoQ10, or olive oil. Pups were then placed in room air from P14 to P21 with no further supplementation. Terminal ileum was assessed for IH-induced injury and inflammatory biomarkers. Neonatal IH induced severe damage consistent with NEC, and was associated with oxidative stress and elevations in PGE2, PGF2α, TxB2, NOS-2 and TLR-4, effects that were ameliorated with nGSH and combination CoQ10+fish oil. Early postnatal supplementation with antioxidants and/or fish oil during neonatal IH may be favorable for preserving gut integrity and reducing oxidative injury.

Effects of omega 3 polyunsaturated fatty acids, antioxidants, and/or non-steroidal inflammatory drugs in the brain of neonatal rats exposed to intermittent hypoxia.

Preterm infants experience frequent arterial oxygen desaturations during oxygen therapy, or intermittent hypoxia (IH). Neonatal IH increases oxidative distress which contributes to neuroinflammation and brain injury. We tested the hypotheses that exposure to neonatal IH is detrimental to the immature brain and that early supplementation with antioxidants and/or omega 3 polyunsaturated fatty acids (n-3 PUFAs) combined with non-steroidal anti-inflammatory drugs (NSAIDs) is protective. Newborn rats were exposed to brief hypoxia (12% O2 ) during hyperoxia (50% O2 ) from the first day of life (P0) until P14 during which they received daily oral supplementation with antioxidants, namely coenzyme Q10 (CoQ10) or glutathione nanoparticles (nGSH), n-3 PUFAs and/or topical ocular ketorolac. Placebo controls received daily oral olive oil and topical ocular saline. Room air (RA) littermates remained in 21% O2 from birth to P21 with all treatments identical. At P14 animals were allowed to recover in RA until P21 with no further treatment. Whole brains were harvested for histopathology and morphometric analyses, and assessed for biomarkers of oxidative stress and inflammation, as well as myelin injury. Neonatal IH resulted in higher brain/body weight ratios, an effect that was reversed with n-3 PUFAs and n-3 PUFAs+CoQ10 with or without ketorolac. Neonatal IH was also associated with hemorrhage, oxidative stress, and elevations in inflammatory prostanoids. Supplementation with n-3 PUFAs and nGSH with and without ketorolac were most beneficial for myelin growth and integrity when administered in RA. However, the benefit of n-3 PUFAs was significantly curtailed in neonatal IH. Neonatal IH during a critical time of brain development causes inflammation and oxidative injury. Loss of therapeutic benefits of n-3 PUFAs suggest its susceptibility to oxidation in neonatal IH and therefore indicate that co-administration with antioxidants may be necessary to sustain its efficacy.

Pharmacodynamic Effects of Standard versus High Caffeine Doses in the Developing Brain of Neonatal Rats Exposed to Intermittent Hypoxia.

(1) Background: Caffeine citrate, at standard doses, is effective for reducing the incidence of apnea of prematurity (AOP) and may confer neuroprotection and decrease neonatal morbidities in extremely low gestational age neonates (ELGANs) requiring oxygen therapy. We tested the hypothesis that high-dose caffeine (HiC) has no adverse effects on the neonatal brain. (2) Methods: Newborn rat pups were randomized to room air (RA), hyperoxia (Hx) or neonatal intermittent hypoxia (IH), from birth (P0) to P14 during which they received intraperitoneal injections of LoC (20 mg/kg on P0; 5 mg/kg/day on P1-P14), HiC (80 mg/kg; 20 mg/kg), or equivalent volume saline. Blood gases, histopathology, myelin and neuronal integrity, and adenosine receptor reactivity were assessed. (3) Results: Caffeine treatment in Hx influenced blood gases more than treatment in neonatal IH. Exposure to neonatal IH resulted in hemorrhage and higher brain width, particularly in layer 2 of the cerebral cortex. Both caffeine doses increased brain width in RA, but layer 2 was increased only with HiC. HiC decreased oxidative stress more effectively than LoC, and both doses reduced apoptosis biomarkers. In RA, both caffeine doses improved myelination, but the effect was abolished in Hx and neonatal IH. Similarly, both doses inhibited adenosine 1A receptor in all oxygen environments, but adenosine 2A receptor was inhibited only in RA and Hx. (4) Conclusions: Caffeine, even at high doses, when administered in normoxia, can confer neuroprotection, evidenced by reductions in oxidative stress, hypermyelination, and increased Golgi bodies. However, varying oxygen environments, such as Hx or neonatal IH, may alter and modify pharmacodynamic actions of caffeine and may even override the benefits caffeine.

Ocular Versus Oral Propranolol for Prevention and/or Treatment of Oxygen-Induced Retinopathy in a Rat Model.

Purpose: Propranolol, a nonselective B1/B2 adrenoceptor antagonist, promotes the regression of infantile hemangiomas likely through suppression of vascular endothelial growth factor (VEGF), which prompted its use for the prevention of retinopathy of prematurity. We tested the hypothesis that topical ocular propranolol is safe and effective for reducing the severity of oxygen-induced retinopathy (OIR) in the neonatal rat intermittent hypoxia (IH) model. Methods: At birth (P0), rat pups were randomly assigned to room air or neonatal intermittent hypoxia (IH) consisting of 50% O2 with brief episodes of hypoxia (12% O2) from P0 to P14, during which they received a single daily dose of oral propranolol (1 mg/kg/day in 50 µL in sterile normal saline) or topical ocular propranolol (0.2% in 10 µL in normal saline) from P5 to P14. Placebo-controlled littermates received 50 µL oral or 10 µL topical ocular sterile normal saline. Retinal vascular and astrocyte integrity; retinal histopathology and morphometry; and angiogenesis biomarkers were determined. Results: Topical ocular propranolol improved retinal vascular damage and preserved the astrocytic template, but did not completely prevent OIR. The beneficial effects of propranolol were associated with reduced ocular VEGF and increased endogenous soluble inhibitor, sVEGFR-1, when administered topically. Conclusions: Propranolol failed to completely prevent severe OIR, however, it prevented astrocyte degeneration resulting from neonatal IH-induced damage. We conclude that the mechanisms of propranolol's beneficial effects in neonatal IH may involve in part, astrocyte preservation.

Acute and chronic effects of intravitreal bevacizumab on lung biomarkers of angiogenesis in the rat exposed to neonatal intermittent hypoxia.

PURPOSE/AIM: Intravitreal bevacizumab (Avastin) is an irreversible vascular endothelial growth factor (VEGF) inhibitor used to treat severe retinopathy of prematurity (ROP) in extremely low gestational age neonates (ELGANs). ELGANs who are at the highest risk for developing severe ROP often experience brief intermittent hypoxia (IH) episodes which may cause oxidative damage. We tested the hypothesis that intravitreal Avastin leaks into the systemic circulation during exposure to IH and has adverse effects on biomarkers of pulmonary microvascular maturation, thus leading to pulmonary hemorrhage and long-term pulmonary sequelae. METHODS: Neonatal rats at postnatal day (PN) 0 (birth) were exposed to either: 1) hyperoxia (50% O2) or 2) neonatal IH (50% O2 with brief episodes of 12% O2) from PN0 to PN14. Room air (RA) littermates served as controls. At PN14, the time of eye opening in rats, a single dose of Avastin (0.125 mg in 5 µL) was injected into the vitreous cavity of the left eyes. A control group received equivalent volume saline. At PN23 and PN45, blood gases, lung-to-body weight ratios, histology, immunofluorescence, and lung biomarkers of angiogenesis were examined. RESULTS: At PN23, Avastin increased lung VEGF, nitric oxide derivatives (NOx), and hypoxia-inducible factor (HIF)1a in the hyperoxia-exposed groups, but decreased soluble VEGFR-1 (sVEGFR-1). At PN45, lungs from animals exposed to neonatal IH and treated with Avastin were severely hemorrhagic with morphologic changes in lung architecture consistent with chronic lung disease. This was associated with higher VEGF and NOx levels, and lower insulin-like growth factor (IGF)-I and sVEGFR-1. CONCLUSIONS: These findings prove our hypothesis that intravitreal Avastin penetrates the blood-ocular barrier in IH and alters lung biomarkers of angiogenesis. Avastin targeting of VEGF could affect normal lung development which may be exaggerated under pathologic conditions such as IH, ultimately leading to vascular permeability, vessel rupture, and pulmonary hemorrhage.

Combination Antioxidant/NSAID Therapies and Oral/Topical Ocular Delivery Modes for Prevention of Oxygen-Induced Retinopathy in a Rat Model.

Given the complexity of oxygen-induced retinopathy (OIR), we tested the hypothesis that combination therapies and modes of administration would synergistically optimize efficacy for prevention of OIR. Newborn rats were exposed to neonatal intermittent hypoxia (IH) from the first day of life (P0) until P14 during which they received: (1) oral glutathione nanoparticles (nGSH) with topical ocular phosphate buffered saline (PBS); (2) nGSH with topical ocular Acuvail (ACV); (3) oral coenzyme Q10 (CoQ10) + ACV; (4) oral omega 3 polyunsaturated fatty acids (n-3 PUFAs) + ACV; (5) CoQ10 + n-3 PUFAs + PBS; or (6) CoQ10 + n-3 PUFAs + ACV. Treated groups raised in room air (RA) served as controls. At P14, pups were placed in RA with no treatment until P21. Retinal vascular pathology, ocular angiogenesis biomarkers, histopathology, and morphometry were determined. All combination treatments in IH resulted in the most beneficial retinal outcomes consistent with suppression of angiogenesis growth factors during reoxygenation/reperfusion and no significant adverse effects on somatic growth. nGSH + PBS also reversed IH-induced retinopathy, but had negative effects on growth. Simultaneously targeting oxidants, inflammation, and poor growth mitigates the damaging effects of neonatal IH on the developing retina. Therapeutic synergy with combination delivery methods enhance individual attributes and simultaneously target multiple pathways involved in complex diseases such as OIR.

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.

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.

SiRNA silencing of VEGF, IGFs, and their receptors in human retinal microvascular endothelial cells.

Vascular endothelial growth factor (VEGF) is a potent mitogen that regulates proliferation, migration, and tube formation of endothelial cells (EC). VEGF has recently become a target for severe retinopathy of prematurity (ROP) therapy. We tested the hypothesis that a specific VEGF isoform and/or receptor acts synergistically with insulin-like growth factor (IGF)-I to alter normal retinal microvascular EC angiogenesis and RNA interference can be used to reverse VEGF effects. We used small interfering RNA (SiRNA) transfection to target VEGF isoforms, IGFs, and their receptors in human retinal microvascular endothelial cells (HRECs). Media was collected at 24 and 48 hours post transfection for measurement of VEGF, sVEGFR-1 and IGF-1 levels; and HRECs were assessed for migration, tube formation, VEGF signaling genes, oxidative stress, and immune-reactivity. At 24 hours post transfection VEGF increased with VEGFR-2; sVEGFR-1 decreased with VEGF165, VEGFR-2, and IGF-1R; and IGF-I increased with VEGF189, VEGFR-1, IGF-2R, IGF+VEGF165, and IGF+VEGF121. IGF-I transfection with each VEGF isoform reduced sphere- forming and migration capacities with robust upregulation of caspase-9, COX-2, MAPK, PKC, and VEGF receptors. At 48 hours, the effects were reversed with a majority of genes downregulated, except with IGF-I and NP-1 transfection. Using RNA interference for targeted inhibition of VEGF isoforms in conjunction with IGF-I may be preferable for suppression of HREC overgrowth in vasoproliferative retinopathies such as ROP.