© 1996 American Heart Association, Inc.
Articles |
Increased Nitric Oxide Synthesis and Action Preclude Choroidal Vasoconstriction to Hyperoxia in Newborn Pigs
the Departments of Pediatrics, Pharmacology, and Ophthalmology, Research Center of Hopital Ste Justine, University of Montreal (P.H., K.G.P., I.L., S.C.), and the Department of Pharmacology and Therapeutics, McGill University (D.R.V.), Montreal, Quebec, Canada.
Correspondence to Dr Sylvain Chemtob, MD, PhD, FRCPC, Research Center, Hopital Ste Justine, 3175, Chemin Cote Ste Catherine, Montreal, Quebec, Canada H3T 1C5. E-mail chemtobs@ere.umontreal.ca.
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Abstract |
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We tested the hypothesis that hyperoxia does not cause adequate constriction of choroidal vessels of the newborn (1 to 5 days old) pig, resulting in increased O2 delivery to the retina, possibly due to excess production and/or effects of vasodilators such as nitric oxide (NO). Hyperoxia (100% O2, 45 minutes) led to a decrease in retinal blood flow (RBF) of both newborn and juvenile (5 to 6 weeks old) pigs and also reduced choroidal blood flow (ChBF) in juvenile but not in newborn pigs; the absence of hyperoxia-induced ChBF response in the newborn was associated with a rise in choroidal O2 delivery. Ibuprofen (prostaglandin G/H synthase inhibitor) and 1,3-dimethyl-2-thiourea (a free radical scavenger) did not modify the choroidal hemodynamic responses to hyperoxia in newborn pigs. However, in newborn animals treated with the NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME), hyperoxia caused a decrease in blood flow and O2 delivery to the choroid. Consistent with these effects of L-NAME, hyperoxia induced an increase in choroidal cGMP in newborn pigs ventilated with 100% O2 and stimulated nitrite production in isolated choroids exposed to hyperoxia from newborn but not juvenile pigs; these effects were inhibited by NOS blockers. Also, both constitutive and inducible NOS activities were higher in choroidal tissues from newborn than from juvenile animals. In addition, the vasorelaxant effect of the NO donor sodium nitroprusside in vitro was also greater on choroids from newborn than from juvenile pigs. Finally, L-NAME prevented the hyperoxia-induced increase in peroxidation products in the choroid of newborns. It is concluded that hyperoxia does not lead to a decrease in blood flow and O2 delivery to the choroid of the newborn because of increased NO synthesis and effects; since the choroid is the main source of O2 supply to the retina, the present data contribute in providing an explanation for the increased susceptibility of the immature neonate to hyperoxia-induced retinopathy.
Key Words: nitric oxide • retina • choroid • cyclooxygenase • hyperoxia
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Introduction |
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Retinopathy of prematurity afflicts up to 80% of premature infants, with serious sequelae such as strabismus, amblyopia, myopia, and in the gravest cases, blindness.1 The most important factor contributing to this vascular disorder appears to be the delivery of excess O2 to the retina.2 3 Premature infants are subject to fluctuating O2 tension, which frequently leads to hyperoxygenation. Hyperoxia can cause excess O2 delivery to the retina provided that ocular vessels do not constrict adequately. In contrast to the adult,4 the newborn does not possess a fully developed autoregulatory control of ocular blood flow5 6 ; this situation would favor retinal hyperoxia as the blood O2 tension increases. However, studies have shown that hyperoxia causes significant constriction of the retinal vasculature in the newborn7 8 as well as in the adult.9 10 11 12 On the other hand, the major source of O2 delivery to the retina, particularly in the newborn, is the choroid,13 14 15 but the effects of hyperoxia on choroidal circulation of the newborn have not been studied. It is thus possible that hyperoxia causes an increase in O2 supply to the retina of the newborn because of increased ChBF secondary to poor vasoconstriction of choroidal vessels.
Autoregulation of ocular blood flow is an important and complex physiological adjustment involving neuronal and hormonal homeostatic mechanisms.13 Prostaglandins and free radicals have been shown to play major roles in the autoregulation of ocular blood flow in newborn animals.5 6 NO is a potent endothelium-derived vasodilator16 but does not seem to be involved in the regulation of RBF during hypoxia, hypotension, and hypercapnia.17 18 On the other hand, inhibition of NOS, predominantly the NOS-3 isoform, decreases ChBF and increases choroidal vascular resistance,17 19 20 and in newborn animals it increases the range of ChBF autoregulation during an acute rise in perfusion pressure.21 It is thus possible that prostanoids, free radicals, and NO contribute to determining the responses of ChBF to hyperoxia. The present studies were undertaken to test the hypothesis that hyperoxia does not cause adequate constriction of choroidal vessels of the newborn, leading to excess O2 delivery to the retina, and that this phenomenon may be due to excess production and/or effects of vasodilators such as NO. Newborn pigs were used because anatomically and to some extent developmentally the retinal circulation of the newborn pig is very similar to that of human infants22 ; for purposes of comparison, juvenile pigs were also used.
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Animals and Surgical Procedures
Newborn (1 to 5 days old) and juvenile (5 to 6 weeks old) pigs were used in this study according to a protocol approved by the Animal Care Committee of the Research Center of Ste Justine Hospital. RBF and ChBF were measured by microspheres as previously described.5 6 21 Briefly, the animals were anesthetized with 2.0% halothane for tracheostomy and catheterization of various blood vessels. The left subclavian artery was catheterized with a polyethylene catheter for the withdrawal of blood samples, including reference samples. A similar catheter was placed into the left ventricle via the right subclavian artery for the injection of radiolabeled microspheres to measure blood flow, and another was placed in the descending thoracic aorta via a femoral artery for continuous blood pressure recording by means of a Statham pressure transducer connected to a Gould multichannel recorder (TA240). A polyethylene catheter was placed in the femoral vein for intravenous administration of drugs. To measure IOP, a 27-gauge butterfly needle attached to a catheter was introduced into the anterior chamber of the eye through the cornea, and the site of entry was sealed with cyanoacrylate glue. Animals were ventilated by means of a Harvard small animal respirator with a gas mixture of 21% O2 and 79% N2. Halothane was discontinued after surgery; animals were maintained on
-chloralose (bolus injection of 50 mg/kg IV followed by infusion of 10 mg·kg-1·h-1) and paralyzed with pancuronium (0.1 mg/kg IV). Body temperature was maintained at 38°C with an overhead lamp, and the animals were allowed to recover from surgery for 2 hours before starting the experiments.
Experimental Protocol and Hemodynamic Measures
RBF, ChBF, and arterial blood gases were measured before as well as 15 and 45 minutes after pigs were ventilated with 100% O2; although values at 15 minutes were nearly the same as those at 45 minutes, to ascertain stability of responses, values at 45 minutes were used for further analysis. Forty animals were randomly assigned to receive intravenously (45 minutes before the first blood flow measure) either saline (1.5 mL) or one of the following agents: 40 mg/kg ibuprofen, a dose previously shown to significantly inhibit prostaglandin synthesis5 23 ; a high dose (750 mg/kg) of DMTU, an effective antioxidant dose24 ; or 1 mg/kg bolus injection followed by a continuous infusion of 50 µg·kg-1·min-1 of L-NAME, a dose that significantly inhibits NOS activity21 25 and is consistent with ID50 and IC50 values for NOS.25 26
RBF and ChBF were determined by the radionuclide-labeled microsphere technique as previously described in detail5 6 ; 
106 microspheres (15 µm diameter) labeled with 141Ce, 95Nb, or 46Sc were injected in a random sequence into the left ventricle. Immediately after the completion of the injection of microspheres, blood samples were withdrawn from the left subclavian artery to determine blood gases, O2 content (ABL 300; Radiometer), and hemoglobin concentrations. After the experiment, animals were killed by intravenous injection of sodium pentobarbital (120 mg/kg); the location of catheters was verified and the eyes were removed. Radioactivity in the retina, choroid, and reference blood samples was counted in a gamma scintillation counter (Cobra II, Canberra Packard). Blood flow (mL·min-1·g-1) was calculated as (cpm/g tissuexreference blood withdrawal rate)/(cpm in the reference blood). O2 delivery to the retina and choroid was calculated as RBF and ChBFxarterial O2 content (µL·min-1·g-1).27 Retinal and choroidal vascular resistances (mm Hg·mL-1·min-1·g-1) were calculated by dividing the ocular perfusion pressure (MBP minus IOP) by RBF and ChBF, respectively.
Assays of cGMP, Hydroperoxides, and 6-Keto PGF1
Seventy additional pigs were surgically prepared and ventilated with 21% O2 or 100% O2 as described above. Immediately after the animals were killed, liquid N2 was poured on each eye. The tissues were then removed and stored for <2 weeks at -80°C.5 6 In an attempt to assess changes in NO release in vivo, we measured its major second-messenger cGMP, which has been shown to be a reliable index of NO activity.26 28 Changes in cGMP were not determined in the retina, because in this tissue cGMP predominantly arises from phototransduction. For each assay, one choroid was homogenized using a tissue grinder (30 000 rpm for 30 seconds; Omni 2000) in a buffer (pH 7.4) containing (in mmol/L) Tris-HCl 10, MgCl2 7.5, EGTA 0.5, 0.02% acetylsalicylic acid, 1,4-dithiothreitol 1, benzamidine 1, and 3-isobutyl-1-methylxanthine 0.5 and then centrifuged for 10 minutes at 1000g. cGMP in the supernatant was measured by radioimmunoassay with a commercial kit (Amersham); the efficiency of recovery was >90%.
For the assay of hydroperoxides, the retinas and choroids were thawed on ice and suspended in a cold buffer (pH 7.4) of the following composition: 5 mmol/L Tris-HCL, 0.67 mmol/L acetylsalicylic acid, 0.5 mmol/L EGTA, and 45 µmol/L BHT. Tissues were homogenized and centrifuged at 1000g for 10 minutes to remove undisrupted cells and nuclei. The supernatant was used for the assay. Hydroperoxides were determined by the oxidation of 250 µmol/L FeCl2 under acidic conditions (H2SO4, pH 2 to 3) in the presence of 4 mmol/L BHT and 100 µmol/L xylenol orange. Standard curves were obtained with t-butyl-hydroperoxide and the absorption was measured at 560 nm6 23 ; the interassay variability was <3.5%.
One choroid for each assay of 6-keto-PGF1 was homogenized in cold buffer (pH 7.4) containing 5 mmol/L Tris-HCl, 0.02% acetylsalicylic acid, and 1 mmol/L EDTA, pH 7.4. The homogenate was centrifuged at 1000g for 10 minutes to remove undisrupted cells and nuclei. The supernatant was rehomogenized and then centrifuged at 50 000g for 30 minutes at 4°C to remove membranes and to enhance extraction of prostanoids on octadecylsilyl silica columns, as previously reported.6 23 6-Keto-PGF1 was measured by radioimmunoassay technique; efficacy of recovery was >96% and interassay variability <2.5%.
NO and cGMP Synthesis by the Choroid In Vitro
NO production by the choroid was measured indirectly by determining the formation of its stable oxidation product, nitrite, using a modification of Wu's method29 ; this method allows conversion of nitrate back to nitrite, which enhances reliability of the latter. Intact choroids were suspended in 1000 µL Krebs' buffer of the following composition (mmol/L): NaCl 120, KCl 4.5, CaCl2 2.5, MgSO4 1.0, NaHCO3 27, KH2PO4 1.0, and glucose 10, in the presence of L-arginine 0.2. Tissues were preincubated for 15 minutes at 37°C and oxygenated with 12% (PO2=70 to 80 mm Hg) or 95% (PO2=490 to 640 mm Hg) O2; these O2 concentrations compared with those obtained in vivo in 21% and 100% O2, respectively. The equilibration period was established by measures of PO2 in the buffer. Aliquots of 100 µL of the buffer were collected over a 15-minute incubation to measure production of nitrite; the choroid was stored at -80°C for the measurement of cGMP, as described above. The buffer samples were added to 20 µL of NADPH and 80 µL of a mixture containing nitrate reductase (80 U/L), glucose-6-phosphate dehydrogenase (160 U/L), and glucose-6-phosphate (500 µmol/L). The reaction time was 45 minutes at 20°C. The components of Griess reagent [200 µL of 0.1% N-(1-naphthyl)ethylenediamine HCl followed by 200 µL of 1% sulfanilamide in 5% phosphoric acid] were then added. After the incubation of samples for 10 minutes at 20°C, the absorbance was read by spectrophotometry (Beckman DU-600) at 540 nm. Standard curves were obtained with sodium nitrite and had an interassay variability of <5%.
NOS Activity
NOS activity was determined essentially as described in the literature.30 Briefly, 0.5 to 1.0 g porcine choroid tissue was collected from three newborn and two juvenile pigs and homogenized in 5 mL ice-cold homogenization buffer (50 mmol/L Tris-HCl, pH 7.5, 1 mmol/L EDTA, 1 mmol/L 1,4-dithiothreitol, 5 mmol/L glucose, 1 mmol/L PMSF, and 20 µg/mL each of aprotinin, leupeptin, and soybean trypsin inhibitor) using a Polytron tissue grinder. The homogenate was centrifuged at 12 000g for 15 minutes and the protein content of the supernatant determined by the dye-binding method, using BSA as the standard. An aliquot of the supernatant (100 to 200 µg protein) was incubated in the presence or absence of 1 mmol/L L-NA in 200 µL incubation buffer (in mmol/L: HEPES 50, pH 7.5, 1,4-dithiothreitol 1, EDTA 1, and CaCl2 1.25) containing 0.1 mmol/L L-[3H]arginine (1 µCi of 91 Ci/mmol specific activity), 1 mmol/L NADPH, 15 µmol/L 6R-tetrahydrobiopterin, 1 µmol/L FAD, and 1 µmol/L calmodulin for 10 minutes at 37°C. The incubations were stopped by adding 1 mL ice-cold 100 mmol/L HEPES (pH 5.5) buffer containing 10 mmol/L EGTA and 500 mg Dowex AG-50W-X8 (counterion Na+) cation exchange resin, and the preparation was immediately centrifuged at 12 000g for 20 minutes. Radioactivity in the L-[3H]citrulline–containing supernatant was counted. To determine iNOS activity, EGTA (10 mmol/L) was added to the incubation buffer. Total NOS activity was calculated as the L-NA–sensitive formation of L-[3H]citrulline. cNOS activity was obtained by subtracting iNOS activity from the total NOS activity.
Choroidal Vessel Responses In Vitro
Eyes were removed from the newborn and juvenile pigs and placed in Krebs' buffer of the following composition (mmol/L): NaCl 120, KCl 4.5, CaCl2 2.5, MgSO4 1.0, NaHCO3 27, KH2PO4 1.0, sodium edetate 0.01, and glucose 10, to which 1.5 U/mL heparin was added. Eyecups were prepared to study the response of the relatively undisturbed vasculature of the choroid as previously described in detail.21 31 Briefly, a circular incision was made 3 to 4 mm posterior to the ora serrata, and the anterior segment and vitreous body were removed. The retinal pigment epithelium was removed to reveal the choroidal vasculature. The eyecups were fixed with pins to a wax base in a 20-mL tissue bath containing Krebs' buffer equilibrated with a mixture of 12% O2, 5% CO2, and the balance N2 and maintained at 37°C and pH 7.35 to 7.45. Preparations were allowed to equilibrate for 30 to 45 minutes, during which time they were washed with fresh buffer every 15 minutes. Segments of choroidal vessels measuring 100 to 200 µm in diameter, reported to be important in blood flow autoregulation,32 were randomly selected. The outer vessel diameter was recorded with a video camera mounted on a dissecting microscope (Zeiss M-400). The digital images were analyzed using Sigma Scan software (Jandel Scientific). Effects of equilibration of the buffer with 12% and 95% O2 on vessel diameter were studied in the presence or absence of 1 mmol/L L-NA. For purposes of comparison, effects of the thromboxane A2 analogue U46619 and PMA on choroidal vessel diameter were determined. To determine the concentration-dependent vasorelaxant response to the NO donor sodium nitroprusside (10-12 to 10-5 mol/L) and carbaprostacyclin, choroidal vessels were precontracted with U46619 (100 nmol/L, 50% of maximal contraction). Each measurement was repeated three times and had a variability of <1%.
Chemicals
Radionuclide microspheres were purchased from New England Nuclear. cGMP radioimmunoassay kits and L-[3H]arginine were obtained from Amersham; prostaglandins and prostaglandin antisera were obtained from Advanced Magnetics. Ibuprofen, DMTU, PMA, L-NAME, and L-NA were purchased from Sigma Chemical Company. U-46619 and carbaprostacyclin were purchased from Cayman Chemicals. All other solvents and chemicals were purchased from Fisher Scientific Chemical Co.
Statistics
Data were analyzed by three-way ANOVA, factoring for O2 concentration, age group, and treatment, and by comparison among mean tests. Statistical significance was set at P<.05.
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Effects of Hyperoxia on MBP, Heart Rate, IOP, and Blood Gases
Hemodynamic and arterial blood gas values in newborn pigs at 21% O2 (n=7) were as follows: MBP (mm Hg), 74±3; heart rate (bpm), 206±14; IOP (mm Hg), 11±1; blood pH, 7.43±0.03; PCO2 (mm Hg), 41±1; PO2 (mm Hg), 110±4; and O2 content (vol%), 9.9±1. Ventilation with 100% O2 significantly (P<.05) increased PO2 to 468±20 and O2 content to 14.4±1.1 but had no significant effect on other variables. Ibuprofen (n=8) and DMTU (n=6) did not affect any of the variables. L-NAME significantly increased MBP from 74±3 to 94±5 at 21% O2 and from 80±3 to 93±5 at 100% O2 (n=6) but did not affect other parameters, including IOP.
Hemodynamic and arterial blood gas variables in juvenile pigs at 21% O2 (n=7) were as follows: MBP, 83±5; heart rate, 245±21; IOP, 16±3; blood pH, 7.41±0.02; PCO2, 41±2; PO2, 108±3; and O2 content, 12.3±1.5. Ventilation with 100% O2 increased PO2 to 485±20 mm Hg and O2 content to 16.2±1.4 vol% (P<.05) without significantly affecting other variables. Injection of L-NAME (n=6) increased MBP from 83±5 to 119±14 during normoxia and from 78±4 to 126±16 under hyperoxia but did not affect any other parameter.
Effects of Hyperoxia on RBF and ChBF
Increasing inhaled O2 from 21% to 100% decreased RBF on average by 44% and 35% in newborn and juvenile pigs, respectively (Fig 1c and 1d
). RBF response to hyperoxia was not modified by ibuprofen, DMTU, or L-NAME in newborn pigs or by L-NAME in juvenile animals; effects of ibuprofen and DMTU were not studied in juvenile pigs.
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, 1.5 mL, n=7), ibuprofen (
, 40 mg/kg, n=8), DMTU (
, 750 mg/kg, n=6), or L-NAME (
, 1 mg/kg followed by 50 µg·kg-1·min-1, n=6). Juvenile pigs (b and d) were treated intravenously with saline (
P<.05 compared with corresponding value in saline-treated animals.
Ventilation with 100% O2 decreased ChBF on average by 35% in juvenile animals (Fig 1b
) but produced no significant effects on ChBF in newborns (Fig 1a). Ibuprofen and DMTU did not affect ChBF response to hyperoxia (Fig 1a). However, L-NAME decreased ChBF in newborn and juvenile pigs during both normoxia and hyperoxia. Moreover, in contrast to saline-treated newborns, L-NAME–treated newborn pigs exhibited a decrease in ChBF in response to hyperoxia, as seen in juvenile pigs (Fig 1a and 1b).
Effects of Hyperoxia on Ocular Vascular Resistance and Oxygen Delivery
Changes in retinal and choroidal vascular resistance were consistent with those for ocular blood flow. Hyperoxia caused an increase in retinal and choroidal vascular resistance in juvenile pigs but only in retinal vascular resistance in newborn animals; these effects of hyperoxia were not significantly altered by ibuprofen, DMTU, or L-NAME (Table).
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Changes in O2 delivery to the retina and choroid paralleled those in blood flow. Hyperoxia decreased O2 delivery to the retina of both newborn and juvenile pigs and to the choroid of juvenile pigs only (Table). Ibuprofen, DMTU, and L-NAME did not affect O2 delivery to the retina. In saline-treated newborn pigs, hyperoxia caused an increase in O2 delivery to the choroid. In contrast, in newborn animals treated with L-NAME, there was a decrease in choroidal O2 delivery, which reduced to a further extent in response to hyperoxia.
Effects of In Vivo Exposure to Hyperoxia on Ocular Tissue Levels of cGMP, Hydroperoxides, and 6-Keto-PGF1
The cGMP levels (pmol/mg protein) in the choroid of newborn pigs increased significantly, from 3.3±0.2 in 21% O2 to 5.2±0.4 in 100% O2, and were decreased after treatment with L-NAME (2.1±0.2, P<.05). Levels of cGMP in newborns were also higher than those in juvenile animals, in which they did not increase with hyperoxia (1.8±0.2 in 21% O2 and 1.9±0.2 in 100% O2).
Exposure of newborn pigs to 100% O2 increased hydroperoxides (nmol/mg protein) in the choroid from 12.5±3.3 to 36.3±9.7 and in the retina from 24.0±3.3 to 44.3±7.1; no changes were seen in juvenile tissues. DMTU markedly reduced hydroperoxides to barely detectable levels, and L-NAME prevented hyperoxia-induced increase in hydroperoxides in newborn tissues. Choroidal 6-keto-PGF1 (pmol/mg protein) levels did not change in response to hyperoxia in newborn animals: 43±3 at 21% O2 and 39±9 during hyperoxia; ibuprofen significantly reduced levels of 6-keto-PGF1 to 12±2 during normoxia and to 15±4 during hyperoxia.
In Vitro Production of cGMP and Nitrite by the Choroid
An attempt to examine whether hyperoxia could stimulate NO production in the newborn was made by exposing isolated choroidal tissue to increased O2 tension. Equilibration of the buffer with 95% O2 resulted in a slight increase in cGMP production in the choroid of newborns over values at 12% O2 (Fig 2); these changes were not observed in tissues of juveniles, which also exhibited lower cGMP formation (Fig 2). L-NA reduced cGMP generation and inhibited its increase in choroids of newborns.
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The formation of nitrite by the choroidal tissues from newborns increased after equilibration with 95% O2, and these levels were higher than the corresponding values in choroids of juvenile pigs (Fig 2). L-NA prevented the hyperoxia-induced increase in nitrite production seen in newborns.
NOS Activity in Choroid
Most of the NOS activity in the choroid was contributed by cNOS; iNOS accounted for 15% of the total in the newborn choroid and was barely detectable in the juvenile choroid (Fig 3). Both the cNOS and iNOS activities in the choroid of newborn pigs were significantly higher than in the choroid of juvenile pigs.
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Effects of Increased Oxygen Concentration and Other Vasoactive Agents on Response of the Isolated Choroid Vessels
An increase in O2 concentration of the buffer from 12% to 95% caused a significantly greater constriction of choroidal vessels from juvenile pigs than of vessels from newborn animals (Fig 4a). However, newborn tissues treated with the NOS blocker L-NA constricted more to 95% O2 than untreated choroidal vasculature. Maximal vasoconstrictor effects of U46619 were also less on newborn than juvenile choroids (28±4% and 54±6%, respectively), whereas the effects of PMA were comparable for these two age groups (48±9% and 50±4%, respectively).
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The relaxant effect of the NO donor sodium nitroprusside was more marked on choroidal vessels from newborns than from juveniles (Fig 4b). Likewise, carbaprostacyclin exerted a greater relaxant effect on the choroid of newborn than of juvenile pigs; maximal effects of carbaprostacyclin in newborn and juvenile pigs were 117±10% and 73±4%, respectively.
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Discussion |
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The present study was conducted to test the postulate that hyperoxia does not cause adequate constriction of choroidal vessels of the newborn, leading to excess O2 delivery to the retina, and that this phenomenon may be due to excess production and/or effects of vasodilators. Our findings indeed confirm this hypothesis. The data indicate that, in contrast to juvenile animals, autoregulatory response of the choroidal circulation to hyperoxia in the newborn is practically absent. Increased activity of NOS and greater vasodilator efficacy of NO in newborns seem to contribute significantly to their lack of hyperoxia-induced autoregulation. Since the choroid is the major source of O2 supply to the retina,13 14 15 this inability of the choroidal vasculature to limit O2 delivery due to excess production and efficacy of NO described here in the newborn provides a new and additional explanation for the increased susceptibility of the neonate to hyperoxia-induced retinal injury.
The decrease in RBF caused by hyperoxia in both newborn and juvenile animals is consistent with findings from several other laboratories.7 8 9 10 11 33 Likewise, the decrease in ChBF we observed in juvenile pigs in response to hyperoxia is in accordance with the results of other workers on adult animals.34 35 36 The decreases in ocular blood flow seem to result from increases in vascular resistance (Table). These hemodynamic changes led to reductions in O2 delivery to the retina of newborn and juvenile pigs as well as to the choroid of juvenile but not of newborn animals. On the contrary, hyperoxia did not cause constriction of the choroidal vessels of the newborn, leading to increased choroidal O2 delivery. In agreement with our findings, it has recently been shown that, in contrast to retinal vasculature, morphometric studies of the choroidal microvasculature of newborn dogs exposed to prolonged hyperoxia revealed no reductions in vessel caliber.37 Associated with this increase in O2 delivery in newborn pigs, peroxidation was also stimulated, as reflected by increases in hydroperoxides in both the choroids and the retinas; the increase in hydroperoxides in the retina, despite a reduction in O2 delivery from retinal circulation, is consistent with the fact that the choroid is by far the main source of O2 supply to the retina.13 14 15
The mechanisms of ocular hemodynamic responses to O2 are for the most part unknown; in other vascular beds, prostaglandins and voltage-dependent calcium channels have been variably implicated.38 39 40 On the other hand, response of the ocular vasculature to an increase in perfusion pressure, which is associated with a rise in O2 delivery in the newborn,5 is better characterized. Prostanoids have been implicated in the regulation of retinal vascular functions by many workers.5 31 41 42 During an acute rise in perfusion pressure, prostaglandins have indeed been shown to be released in ocular tissues,5 6 and inhibition of prostaglandin synthesis enhances RBF and ChBF autoregulatory response to increases in perfusion pressure.5 6 Free radicals have also been suggested to contribute significantly to this acute hypertension–induced RBF and ChBF autoregulation.6 However, during exposure to hyperoxia, there was no increase in ocular prostaglandins, and the cyclooxygenase inhibitor ibuprofen, as well as the hydroxyl radical scavenger DMTU,24 43 44 did not modify the effects of hyperoxia on RBF and ChBF in newborn pigs (Fig 1); lack of stimulation of prostaglandin synthesis by hyperoxia in the newborn eye has similarly been demonstrated by other workers.45 Thus, the data suggest that prostanoids and hydroxyl radicals do not play an important role in the responses of ocular circulation to hyperoxia in newborns. On the other hand, elimination of the vasodilator NO by L-NAME reduces the delivery of O2, which mostly arises from the choroid,13 14 15 to the eye and as a result limits peroxidation in the newborn developmentally deficient in antioxidants.46 47 48 The absence of effect of DMTU on ocular blood flow may therefore be explained by the scavenging action of this drug specifically on hydroxyl radicals but not on NO or superoxide,24 43 44 which could have otherwise altered the hemodynamic response to hyperoxia.
The most important finding of this study is that hyperoxia caused a decrease in ChBF (Fig 1) and an increase in choroidal vasoconstriction (Fig 4a) in the juvenile but not in the newborn pigs; this could be due to one or several factors, such as ontogenic differences in NOS activities (Figs 2 and 3), in the effects of hyperoxia on NO release (Fig 2), or in the sensitivity of NOS to O2 (Fig 2 and the Table). The possibility of an increased sensitivity of NOS in the newborn to O2 is suggested by the data showing that O2 caused a greater increase in vascular cGMP and nitrite production by the choroid of the newborn than of the juvenile (Fig 2). This inference is further supported by a relatively larger rise in choroidal vascular resistance by NOS inhibitors in newborn than in juvenile pigs under both normoxic and hyperoxic conditions, despite a greater ability of the juvenile to vasoconstrict in response to hyperoxia (Table and Fig 4).
A contribution of NO to basal ChBF is suggested by the observation that L-NAME decreased basal ChBF in both the newborn and juvenile pigs in the presence of an increase in ocular perfusion pressure. This reduction in ChBF was greater in newborn (52% to 58%) than in juvenile animals (22% to 36%, P<.05; Fig 1), consistent with a greater production and/or efficacy of NO in the newborn. The reduction we observed in ChBF after NOS inhibition in juvenile animals is similar to that described by others.17 19 20 L-NAME (Fig 1) did not affect RBF in response to hyperoxia in newborn or juvenile pigs, as previously reported.17 18 21 More importantly, in the newborn, in addition to reduced basal choroidal circulation, blood flow and O2 delivery to the choroid decreased during hyperoxia after inhibition of NOS by L-NAME. Although the NOS inhibition–induced hypertension might modify autoregulatory response, as demonstrated in adult rats,49 we have shown that in the newborn animal, hypertension per se (produced by obstructing the aortic isthmus), which results in an increase in ocular perfusion pressure up to 146 mm Hg, induced no autoregulatory response of choroidal blood flow.6 21 Thus, the hyperoxia-induced vasoconstriction of choroidal vessels in newborns after NOS inhibition suggests that it is NOS inhibition rather than hypertension that confers choroidal autoregulatory response, apparently by unmasking a vasoconstriction. Finally, although we cannot totally rule out additional effects of NOS inhibitors on systems other than NO, the doses used are consistent with ID50 and IC50 values for NOS,25 26 and the effects of L-NAME observed in juvenile pigs correspond with those reported with the same and other types of NOS inhibitors,17 18 19 20 21 which can be reversed with L-arginine.19 20
The above inference on the role of NO drawn from in vivo studies is also supported by the data showing that the vasoconstrictor response of eyecup choroidal vessels to hyperoxia increased in the presence of L-NA in the newborn (Fig 4); however, hyperoxia still caused less constriction of the choroidal vessels of the newborn than of juvenile pigs. This lesser constriction of the choroidal vessels of the L-NA–treated newborn than of juvenile animals might in part be due to a generally decreased efficacy of constrictors in the newborn.50 This inference is supported by choroidal vasoconstrictor response to the thromboxane analogue U46619; however, constriction elicited by non–receptor-mediated stimuli such as phorbol esters caused similar effects in newborn and juvenile tissues.
Additional evidence for an important role for NO in the control of choroidal circulation of the newborn is provided by data on cGMP and nitrite production by the choroid. (1) Basal levels of cGMP, a major second messenger of NO,51 as well as in vitro production of cGMP and nitrite, were higher in tissues from newborn than juvenile animals and increased during hyperoxia only in newborn choroids (Fig 2). A stimulant effect of O2 on NOS has been reported by others52 ; reasons for lack of this effect in juvenile choroids are not clear but may be related to decreased activation of calcium channels by O2 and in turn endothelial cNOS activity in older subjects.39 53 (2) Inhibition of NOS prevented the hyperoxia-induced increases in cGMP and nitrite synthesis. (3) cNOS and iNOS activities were significantly higher in choroids of newborn than of juvenile pigs (Fig 3); these findings are consistent with a greater production of nitrite in the newborn choroid (Fig 2) and higher levels of NOS activity reported in other newborn tissues.54 55
Finally, a greater relaxant effect of the NO donor sodium nitroprusside was also disclosed in newborn than in juvenile tissues in the present study (Fig 4b). Reasons for this effect are not clear but are perhaps related to developmental changes in relaxation-coupled mechanisms. The greater relaxant effects of both sodium nitroprusside and carbaprostacyclin in the newborn might suggest an expression of greater responsiveness to vasodilators in newborns than in juveniles, since these two agents act mainly via cGMP and cAMP, respectively; similar ontogenic differences in vasorelaxant response to other agents have been observed.56 57 Increased formation of cGMP by NO in newborn tissues is unlikely, as suggested from the relatively smaller production of cGMP than of nitrite in response to 95% O2 (Fig 2); similar absence of ontogenic changes in guanylate cyclase activity has been reported in other tissues.58 On the other hand, an increased efficacy of sodium nitroprusside on the outward rectifier potassium channel of vasculature of the newborn animal59 may explain the greater sodium nitroprusside–induced vasorelaxation we observed. Overall, our data disclose an important role for NO in the choroidal circulation autoregulatory response to hyperoxia in the newborn and indicate that an inhibition of NOS unveils a vasoconstrictor response to hyperoxia in the choroid of newborn pigs. It is of interest that a more important role of the NO-cGMP system in the regulation of tone of other vascular beds in newborns than in adults has also recently been reported.60
In conclusion, the present findings all together reveal that increased NO production and vasodilator effects in newborns prevent a decrease in ChBF during acute episodes of hyperoxia; since the choroid is the major source of O2 to the retina, these effects allow hyperoxygenation of the retina and favor peroxidation. Given the underdeveloped antioxidant systems in the newborn,46 47 48 this inability to limit O2 delivery could contribute to the high susceptibility of the immature neonate to hyperoxia-induced retinopathy. However, although excess NO actions may lead to deleterious effects, a sufficient amount of this agent may be required to avoid irreversible retinal vasoconstriction and vaso-obliteration.61
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Selected Abbreviations and Acronyms |
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-nitro-L-arginine
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Acknowledgments |
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This work was supported by grants from the Medical Research Council of Canada, the Heart and Stroke Foundation of Quebec, the Hospital for Sick Children Foundation, the United Cerebral Palsy Foundation, and the March of Dimes Birth Defects Foundation. Pierre Hardy is a recipient of a fellowship from the Medical Research Council of Canada. Isabelle Lahaie is a recipient of a studentship from the Fonds de la Recherche en Sante du Quebec. We thank Hensy Fernandez for her technical assistance.
Received March 8, 1996; accepted May 22, 1996.
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