Charles L. Cai

Effects of probiotics, prebiotics, and synbiotics on messenger RNA expression of caveolin-1, NOS, and genes regulating oxidative stress in the terminal ileum of formula-fed neonatal rats.

Necrotizing enterocolitis (NEC) afflicts extremely low birth weight neonates, and probiotics reduces its incidence and severity. NO is involved in the pathogenesis of NEC, and caveolin-1 regulates NO signaling. We tested the hypothesis that intestinal caveolin-1 and NOS are deficient in formula-fed neonatal rats and that supplementation with “Florastar Kids” and/or galacto-oligosaccharides and fructo-oligosaccharides preserves caveolin-1 and NOS. At birth (P0), neonatal rat pups were maternally fed or hand-gavaged with or without supplemented formula. Samples from the terminal ileum were analyzed for total NO metabolites, growth factors, and gene expression of caveolin-1, NOS isoforms, and antioxidants. Our data showed that formula feeding with and without supplementation resulted in significant growth restriction. Despite suboptimal nutrition, growth factors involved in intestinal repair and regeneration were increased in the neonatal rat ileum. Caveolin-1, endothelial NOS, and neuronal NOS were simultaneously down-regulated with formula feeding while inducible NOS was up-regulated. Superoxide dismutase and glutathione peroxidase were up-regulated with supplementation. These data provide a probable mechanism for the benefits of supplemented formula for decreasing the severity of NEC by preserving the antioxidant systems.

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.

Comparative effects of probiotics, prebiotics, and synbiotics on growth factors in the large bowel in a rat model of formula-induced bowel inflammation.

Objectives: Supplementation with probiotics has been shown to prevent gastrointestinal damage possibly through preservation of growth factors. We tested the hypothesis that probiotics, prebiotics, or synbiotics supplementation preserves intestinal insulin-like growth factors (IGFs) and epidermal growth factors (EGFs) in formula-fed neonatal rats. Materials and Methods: At birth (postnatal day 0 [P0]), neonatal rat pups (n = 18 pups/group) were either maternally fed or hand-gavaged with formula supplemented with probiotics (Pro-Fed), prebiotics, or synbiotics from P0 to P3. A formula-fed control group received formula without supplementation. At P4, large bowel samples were assessed histologically and assayed for vascular endothelial growth factor (VEGF), soluble VEGF receptor-1, IGF-I, IGF-II, and EGF. Results: All formula-fed groups were severely growth suppressed with comparable mortalities. Moderate preservation of bowel integrity was noted in the Pro-Fed group. In contrast, severe inflammation was seen in all of the other formula groups. This was associated with significant increases in VEGF levels in all of the formula groups (P < 0.05) except the Pre-Fed group. Similar elevations in soluble VEGF receptor-1 (P < 0.05), IGF-I (P < 0.05), and EGF (P < 0.05) were noted, but statistical significance was achieved only in the Pro-Fed group. Conclusions: Induction of IGF-I and EGF with moderate bowel integrity may represent a protective effect of probiotics against formula-induced inflammation. These data, taken collectively, suggest that probiotics may provide more beneficial effects on the developing large bowel than prebiotics and synbiotics.

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.

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.

Ocular Adverse Effects of Intravitreal Bevacizumab Are Potentiated by Intermittent Hypoxia in a Rat Model of Oxygen-Induced Retinopathy

Intravitreal bevacizumab (Avastin) use in preterm infants with retinopathy of prematurity is associated with severe neurological disabilities, suggesting vascular leakage. We examined the hypothesis that intermittent hypoxia (IH) potentiates intravitreal Avastin leakage. Neonatal rats at birth were exposed to IH from birth (P0)–P14. At P14, the time of eye opening in rats, a single dose of Avastin (0.125 mg) was injected intravitreally into the left eye. Animals were placed in room air (RA) until P23 or P45 for recovery (IHR). Hyperoxia-exposed and RA littermates served as oxygen controls, and equivalent volume saline served as the placebo controls. At P23 and P45 ocular angiogenesis, retinal pathology and ocular and systemic biomarkers of angiogenesis were examined. Retinal flatmounts showed poor peripheral vascularization in Avastin-treated and fellow eyes at P23, with numerous punctate hemorrhages and dilated, tortuous vessels with anastomoses at P45 in the rats exposed to IH. These adverse effects were associated with robust increases in systemic VEGF and in both treated and untreated fellow eyes. Histological analysis showed severe damage in the inner plexiform and inner nuclear layers. Exposure of IH/IHR-induced injured retinal microvasculature to anti-VEGF substances can result in vascular leakage and adverse effects in the developing neonate.

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 antioxidants 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.

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.