Journal of Applied Physiology AJP: Cell Physiology
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J Appl Physiol 91: 1655-1662, 2001;
8750-7587/01 $5.00
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Vol. 91, Issue 4, 1655-1662, October 2001

Major role for neuronal NO synthase in curtailing choroidal blood flow autoregulation in newborn pig

P. Hardy1, D. Lamireau1, X. Hou1, I. Dumont1, D. Abran1, A.-M. Nuyt1, D. R. Varma2, and S. Chemtob1,2

1 Departments of Pediatrics, Ophthalmology, and Pharmacology, Research Center of Hôpital Sainte-Justine, Montréal H3T 1C5; and 2 Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec, Canada H3G 1Y6


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We examined whether nitric oxide (NO) generated from neuronal NO synthase (nNOS) contributes to the reduced ability of the newborn to autoregulate retinal blood flow (RBF) and choroidal blood flow (ChBF) during acute rises in perfusion pressure. In newborn pigs (1-2 days old), RBF (measured by microsphere) is autoregulated over a narrow range of perfusion pressure, whereas ChBF is not autoregulated. NG-nitro-L-arginine methyl ester (L-NAME) or specific nNOS inhibitors 7-nitroindazole, 3-bromo-7-nitroindazole, and 1-(2-trifluoromethyl-phenyl)imidazole as well as ganglionic blocker hexamethonium, unveiled a ChBF autoregulation as observed in juvenile (4- to 6-wk old) animals, whereas autoregulation of RBF in the newborn was only enhanced by L-NAME. All NOS inhibitors and hexamethonium prevented the hypertension-induced increase in NO mediator cGMP in the choroid. nNOS mRNA expression and activity were three- to fourfold higher in the choroid of newborn pigs than in tissues of juvenile pigs. It is concluded that increased production of NO from nNOS curtails ChBF autoregulation in the newborn and suggests a role for the autonomic nervous system in this important hemodynamic function, whereas, for RBF autoregulation, endothelial NOS seems to exert a more important contribution in limiting autoregulation.

ocular blood flow; retina; choroid; nitric oxide


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

RETINAL AND CHOROIDAL BLOOD FLOW (RBF and ChBF, respectively) autoregulation allows blood flow to be maintained constant despite fluctuations in systemic blood pressure (BP) ensuring adequate capillary exchange (8). In the newborn pig, in contrast to the adult (28, 29, 39, 40), RBF is autoregulated over a narrow range and ChBF exhibits no autoregulation (13); consequently, during an acute rise in perfusion pressure, RBF and ChBF of the newborn cannot be maintained constant (13, 23, 24), resulting in potentially toxic increased delivery of oxygen to the retina of the immature subject (13, 23, 26). This lack of pressure-induced autoregulation in the newborn is largely ascribed to excess expression, activity, and interactions of vasodilatory mediators including vasorelaxant prostaglandins and, to a large extent, nitric oxide (NO), both of which mask the effects of constrictors (1, 13, 23, 24). But the relative contribution of NO from endothelium compared with that from autonomic nervous system in ocular blood flow autoregulation remains thus far unknown.

NO produced by NO synthase (NOS) is an important mediator in vasomotor function in the retina and the choroid (26, 30, 34, 50). Both isoforms of calcium-dependent constitutive NOS (cNOS), designated as endothelial and neuronal NOS (eNOS and nNOS, respectively), are present in the retina and the choroid. eNOS is found in the retinal and choroidal vascular endothelium (1, 31), whereas nNOS is localized in neurons and Müller cells of the retina (31, 41) and in the nonadrenergic, noncholinergic parasympathetic perivascular nerve fibers from the pterygopalatine ganglion, which richly innervate the choroid (14, 44, 45, 50); the retinal microvasculature lacks autonomic innervation (5), although this vasculature is closely associated with nNOS-containing neurons (41).

NO from eNOS is involved in resting retinal and choroidal circulation of newborn and adult animals (16, 20, 21, 24, 34, 38); eNOS is also activated by shear stress (42). nNOS does not appear to be implicated in basal ocular blood flow regulation (34, 44). nNOS is normally activated during neural stimulation (34, 44, 50), and parasympathetic stimulation induces an nNOS-dependent (34, 44) increase in ChBF (50). Whether this response applies during physiological adaptions, such as autoregulatory oculovascular control, has never been demonstrated.

Inhibition of all NOS activity extends the upper limit of RBF and ChBF autoregulation in the newborn (24). However, the relative role of nNOS in oculovascular autoregulatory response is not known. The purpose of this study was to test the hypothesis that inhibition of nNOS using specific nNOS inhibitors would significantly extend the upper limit of ocular blood flow autoregulation in the newborn; in this process, we also evaluated (by using ganglionic blockade) the contribution of autonomic nervous system activation. Our findings disclose a significant role for nNOS in ChBF, but not RBF, autoregulation of the newborn.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Yorkshire newborn (1-2 days old) and juvenile (4-6 wk old) pigs obtained from Fermes Ménard (L'Ange-Gardien, Québec, Canada) were used in the present study. Newborns were studied within 12-24 h of arrival and juveniles within 3-5 days; animals were maintained at the Research Center facilities at 25°C, 50-70% humidity, and a 12:12-h light-dark cycle (lights on 7:00 AM to 7:00 PM) and fed ad libitum milk (newborns) or pig chow and tap water (juveniles). The experimental protocol was approved by the Animal Care Committee of Hôpital Sainte-Justine Research Center in accordance with the principles of the "Guide to the Care and Use of Experimental Animals" and guidelines of the Canadian Council on Animal Care Research.

Animal preparation for RBF and ChBF studies. Surgical procedures were conducted under 2% halothane general anesthesia as previously described (13, 23, 24). Tracheostomy was performed, and catheters were placed into the left subclavian artery for the withdrawal of blood samples, including reference samples, into the left ventricle through the right subclavian artery for the injection of radiolabeled microspheres, and into the femoral artery for continuous BP recording with a pressure transducer (Staham, Glen Burnie, MD) connected to a multichannel recorder (TA240 Gould, Valley View, OH). A silicone-coated balloon-tipped catheter (Berman Angiocath, 4 or 8 Fr; Arrow, Reading, PA) was placed in the thoracic descending aorta via a femoral artery distal to the extremity of the catheter used for BP recording; inflation of this balloon produced hypertension in the aortic arch. A second balloon-tipped catheter was placed at the root of the aorta via the right common carotid artery, and its inflation produced hypotension in the aortic arch. A polyethylene catheter was placed in the femoral vein for drug administration. To measure intraocular pressure (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. Halothane was discontinued after surgery. Animals were then maintained on alpha -chloralose (50 mg/kg iv bolus injection followed by infusion of 10 mg · kg-1 · h-1) (25, 35) and paralyzed with pancuronium (0.1 mg/kg iv) for rigorous control of blood gases during the study period. Body temperature was maintained at 38°C with an overhead radiant lamp. Animals were allowed to recover from the surgery for 2 h before the experiments were started.

Measurements of RBF and ChBF. Blood flow was determined using the radionuclide-labeled microsphere technique as previously described (13, 23, 24). Approximately 106 microspheres (15 µm diameter) labeled with 141Ce, 95Nb, 46Sc, or 113Sn (Dupont NEN) were injected in a random sequence into the left ventricle. Reference blood samples were collected during the following 70 s. After each injection of microspheres, blood samples were withdrawn to determine blood gases, oxygen content, and hemoglobin concentrations (ABL 300; Radiometer, Copenhagen, Denmark). These remained within normal limits. After the experiment, pigs were killed with pentobarbital sodium (120 mg/kg iv), the location of catheters was verified, and the eyes were removed. The eyes were weighed, the anterior structures of the eye and the vitreous were gently removed, and the retina and choroid were separated. Radioactivity in the retina, choroid, and reference blood samples were counted in a gamma scintillation counter (Cobra II; Canberra Packard, Meriden, CT). Blood flow (ml · min-1 · g-1) was calculated as [counts per min (cpm)/g tissue × reference blood withdrawal rate]/(cpm in the reference blood). Vascular resistances (mmHg · ml-1 · min-1 · g-1) in the retina and choroid were calculated by dividing the ocular perfusion pressure (OPP) (mean BP - IOP) by RBF and ChBF, respectively.

Ocular blood flow autoregulation was studied as previously described (13, 23, 24). Basal RBF and ChBF were measured 1 h after drug injection, and, 10 min later, one of the two balloon-tipped catheters was inflated to produce hypotension or hypertension. Once steady-state BP was achieved (within 30-40 s of balloon inflation), blood flow was determined again as described above. The balloon was deflated immediately after the blood flow measurements, and animals were allowed to recover for 40 min. At the end of this recovery period, a second baseline blood flow was measured, and, 10 min later, final measurements of RBF and ChBF were made after the other balloon-tipped catheter was inflated. Each animal was subjected to one hypotensive and one hypertensive episode in a random order; for each treatment group of animals, OPP was scaled at intervals of 5 ± 1.5 mmHg to cover a range of 14-146 mmHg.

To specifically study the upper limit of ocular blood flow autoregulation, blood flow was measured in separate animals at preselected mean arterial BP (MBP) values at and above the upper limit of the RBF autoregulation range of the newborn pig (17, 24). MBP was increased stepwise to OPP values of 90, 105, and 125 mmHg [all above the 80 mmHg upper limit of RBF autoregulation in newborn pig (13)]; these values varied by 5 mmHg.

Treatments. Newborn pigs were randomly assigned to receive saline (1.5 ml iv; n = 12) or one of the following NOS inhibitors (bolus of 1 mg/kg iv followed by an infusion of 50 µg · kg-1 · min-1): nonselective NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; n = 11) (24, 38) and nNOS inhibitors 7-nitroindazole monosodium salt (7-NINA) (n = 11) (6, 32), 3-bromo-7-nitroindazole (n = 3) (9), and 1-(2-trifluoromethyl-phenyl)imidazole (TRIM; n = 3) (22). Doses used corresponded to NOS inhibitory activities (6, 22, 38). Hexamethonium, an autonomic ganglionic blocker (1 mg/kg iv; n = 3), was also administered to assess the contribution of the autonomic nervous system on RBF and ChBF autoregulation in newborn animals (12). Juvenile pigs received saline (1.5 ml iv; n = 3) to serve for comparison.

cGMP radioimmunoassay. To assess NO generation in choroid in response to acute hypertension, its major mediator cGMP (15) was measured. Forty-two additional pigs were surgically prepared as described above and kept in normotension or subjected to increased perfusion pressure (OPP = 110 ± 5 mmHg). Immediately after the 2-min induction of hypertension in newborn and juvenile pigs, the pigs were killed and liquid N2 was poured on each eye; choroids were removed and stored for <2 wk at -80°C (26). Choroids were homogenized using a tissue grinder (30,000 rpm for 30 s; Omni 2000, Waterbury, CT) in a buffer (pH 7.4) containing (in mM) 10 Tris · HCl, 7.5 MgCl2, 0.5 EGTA, 1 1,4-dithiothreitol, 1 benzamidine, 0.02% acetylsalicylic acid, and 3-isobutyl-1-methylxanthine 0.5 and then centrifuged for 10 min at 1,000 g. cGMP in the supernatant was measured by radioimmunoassay with a commercial kit (Amersham); the efficiency of recovery was >90%.

eNOS and nNOS RNase protection assays. The partial cDNAs encoding newborn and juvenile pigs' eNOS, nNOS, and destrin mRNAs (33) were synthesized by RT-PCR from porcine choroid total RNA using gene-specific primer sets as described previously (17, 18, 37). The primer pairs for porcine eNOS were 5'-GCT TTT CCC TGC AGG AGC GAC-3' and 5'-GCC AGT CTC TGC AGA CTC TGG-3' (52). The primer pairs for porcine nNOS were 5'-GGG GGA TCC ARG ART AYG ARG ART GGA ART GG-3' and 5'-GGG GAA TTC GAT RTC RAA YTG CGY TGY TGC CA-3' (18). The primer pairs for porcine destrin were 5'-ATG ATG CAA GCT TTG AAA CC-3' and 5'-GGA AGC TTT CGA TCT GTG G-3'. The amplified products (0.4 kb) were digested with appropriate restriction enzyme (underlined sequences in the primers denote the restriction sites) and cloned into plasmid vector pGEM-4Z (Promega).

32P-labeled cRNA probes for eNOS, nNOS, and destrin were prepared using an in vitro transcription kit (Promega). Aliquots of the total RNAs from choroid were subjected to RNase protection assays according to a published protocol with minor modifications (10). Briefly, 60 µg of total RNA were mixed with 105 cpm of eNOS, nNOS, and destrin probes in 20 µl of hybridization buffer (80% deionized formamide, 40 mM PIPES, pH 6.8, 1 mM EDTA, and 0.4 M NaCl), denatured at 90°C for 5 min, and incubated overnight at 50°C. The RNA hybrids were digested with RNase A (10 µg/ml) and RNase T1 (200 U/ml) in 200 µl of digestion buffer (10 mM Tris · HCl, pH 7.5, 5 mM EDTA, and 0.3 M NaCl) for 30 min at 25°C, followed by precipitation of protected fragments (10). The protected RNA fragments were resolved on urea-6% polyacrylamide gels, and the bands were visualized by phosphorimaging (Molecular Dynamics) and quantified densitometrically.

NOS activity. NOS activity in newborn and juvenile porcine choroids was determined by the conversion of L-arginine to L-citrulline as described in detail (18, 26). Choroidal tissues were homogenized in 5 ml of ice-cold homogenization buffer (50 mM Tris · HCl, pH 7.5, 1 mM EDTA, 1 mM 1,4-dithiothreitol, 5 mM glucose, 1 mM phenylmethylsulfonyl fluoride, 3 µM aprotinin, 40 mM leupeptin, and 1 mM soybean trypsin inhibitor). The homogenate was centrifuged at 12,000 g for 15 min, and the protein content of the supernatant was determined by the Bio-Rad dye-binding assay. An aliquot of the supernatant (100-200 µg protein) was incubated in the absence or presence of Nomega -nitro-L-arginine (L-NA; 1 mM) or 7-NINA (100 µM) in incubation buffer [in mM: 50 HEPES (pH 7.5), 1 1,4-dithiothreitol, 1 EDTA, and 1.25 CaCl2] with 0.1 mM L-arginine [containing 1 µCi L-[3H]arginine- (91 µCi/mmol specific activity)], 1 mM NADPH, 15 µM 6R-tetrahydrobiopterin, 1 µM FAD, and 1 µM calmodulin for 10 min at 37°C. The reaction was terminated by the addition of 1 ml of ice-cold 100 mM HEPES buffer (pH 5.5), containing 10 mM EGTA and 500 mg Dowex AG-50W-X8 cation exchange resin and followed by a centrifugation at 10,000 g for 20 min. Radioactivity in the L-[3H]citrulline-containing supernatant was counted. Total NOS and nNOS activities were measured, respectively, from the L-NA- and 7-NINA-sensitive production of L-[3H]citrulline from L-[3H]arginine. Inducible NOS (iNOS) activity was determined by adding EGTA (10 mM) to the incubation buffer. cNOS activity was obtained by subtracting iNOS activity from total L-NA-sensitive NOS activity. 7-NINA-sensitive NOS activity was measured as total NOS activity minus that after addition of 7-NINA.

Chemicals. The following products were purchased: L-NAME, hexamethonium, L-NA, soybean trypsin inhibitor (type II-S), phenylmethylsulfonyl, beta -mercaptoethanol, 1,4-dithiothreitol, HEPES, EDTA, EGTA, L-arginine, NADPH, FAD, and (6R)-tetrahydrobiopterin (Sigma Chemical-Aldrich, Oakville, Ontario); 7-NINA, 3-bromo-7-nitroindazole and TRIM (Tocris Cookson, Allwin, MO); radiolabeled microspheres (DuPont-NEN, Boston, MA); Dowex AG-50W-X8 resin and protein assay (Bio-Rad, Mississauga, Ontario); L-[3H]arginine- and cGMP assay kits (Amersham, Mississauga, Ontario); aprotinin and leupeptin (Boehringer-Mannheim, Montreal, Quebec); RNase protection assay kit (Promega, Madison, WI). All other chemicals came from Fisher (Montreal, Quebec).

Statistical analysis. RBF and ChBF data were analyzed by regression analysis as previously described in detail (23). For linear and nonlinear correlation, the Pearson product-moment coefficient (r) and the Kendall coefficient of rank correlation (tau ) were calculated. The best-fit line for the relationship between blood flow and perfusion pressure was determined by using the method of least squares of a polynomial regression analysis and by calculating the coefficient of determination (R2) (23). In addition, the relationship of blood flow to perfusion pressure was examined by the method of Lowess; smoothing and, based on these curves, separate linear regressions were performed by using the random effect model for longitudinal data. Linear regressions were compared by regression equality test with the use of the method of least squares. Other data were analyzed by ANOVA, factoring for treatment and age group, and by a comparison among means test (Tukey-Kramer method) or by Student's t-test. Statistical significance was set at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Stability of preparations. Arterial pH, PO2, and PCO2 remained stable throughout the course of the experiments (Table 1). RBF and ChBF did not differ between the first and second baselines. L-NAME caused an expected increase in basal MBP and corresponding OPP (augmentation by 31 ± 5%) and a decrease in ChBF (58-64%) without changes on resting RBF (24). 7-NINA, 3-bromo-7-nitroindazole and TRIM did not affect resting MBP, OPP, RBF, and ChBF as anticipated (32, 34, 45). Ganglionic blockade with hexamethonium caused a small reduction in baseline MBP and OPP (8 ± 2%).

                              
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  		Table 1.   Arterial blood pH and gases, IOP, mean arterial BP, OPP, RBF, and ChBF at baseline and during hypotension and hypertension in newborn pigs treated with saline, L-NAME, or 7-NINA

Effects of L-NAME and 7-NINA on RBF and ChBF autoregulation. RBF and ChBF plotted as a function of OPP is shown in Fig. 1. In saline-treated animals, RBF and ChBF correlated nonlinearly with OPP (tau  = 0.60 and 0.72, respectively, P < 0.01; Fig. 1A). RBF was constant between 30 and 80 mmHg of OPP (r = 0.20, P > 0.1, based on polynomial and Lowess curves) and varied with OPP above and below this range (r = 0.82, P < 0.01 and 0.67, P < 0.05, respectively). ChBF increased as a function of OPP over the entire range of OPP studied (r =0.95, P < 0.01). Treatment with L-NAME, a nonspecific NOS inhibitor, significantly broadened the range of RBF autoregulation up to 146 mmHg (the maximum OPP studied) and allowed ChBF to be maintained constant between 30 and 146 mmHg such that the RBF and ChBF relations to OPP differed significantly from those in saline-treated animals (P < 0.01) by regression equality test (Fig. 1B). Treatment with the preferential nNOS inhibitor 7-NINA uncovered a ChBF autoregulation at OPP >30 mmHg compared with saline-treated animals (P < 0.01) but had no effect on RBF autoregulation (tau  = 0.60, P < 0.05; Fig. 1C). L-NAME and 7-NINA did not affect the RBF and ChBF response to OPP <30 mmHg (Fig. 1, B and C).


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  		Fig. 1.   Retinal and choroidal blood flow (RBF and ChBF, respectively) autoregulation as a function of ocular perfusion pressure (OPP) in newborn pigs treated with either saline (1 mg/kg iv) (A), nitrogen oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME; B), or 7-nitroindazole monosodium salt (7-NINA; C) (1 mg/kg iv followed by 50 µg · kg-1 · min-1) (n = 8 for each group). Each animal was subjected to one hypotensive and one hypertensive episode to cover, for each treatment group, a range of OPP from 5 to 146 mmHg. In saline-treated animals, the best-fit regression was a third-order polynomial for RBF (R2 = 0.88, P < 0.05) and a linear (single-order) regression for ChBF (R2 = 0.78, P < 0.05) (A). In L-NAME-treated pigs, a second-order polynomial regression best fit the points for RBF and ChBF (R2 = 0.73 and 0.81, respectively, P < 0.05) (B). In 7-NINA-treated pigs, a third-order polynomial regression best fit the points for RBF (R2 = 0.91, P < 0.05), and a second-order polynomial regression fit best for ChBF (R2 = 0.75, P < 0.05) (C).

Effects of L-NAME, 7-NINA, 3-bromo-7-nitroindazole, and TRIM on the upper limit of ChBF autoregulation. Because ChBF, not RBF, autoregulation was affected by both L-NAME and 7-NINA (Fig. 1), we focused on the role nNOS specifically had on perfusion pressure-induced ChBF autoregulation. In saline-treated newborn animals, ChBF increased linearly with OPP >80 mmHg (r = 0.65-0.91, P < 0.01; Fig. 2A). L-NAME, 7-NINA, 3-bromo-7-nitroindazole, and TRIM (Figs. 2, B-E) prevented the change in ChBF as a function of OPP (L-NAME: r = 0.12-0.31, P > 0.1; 7-NINA: r = 0.08-0.18, P > 0.1; 3-bromo-7-nitroindazole: r = 0.11-0.22, P > 0.1; and TRIM: r = 0.20-0.34, P > 0.1), as seen in juvenile pigs (r = 0.22-0.36, P > 0.5; Fig. 2F). Regressions for saline-treated newborn animals differed significantly from those treated with L-NAME, 7-NINA, 3-bromo-7-nitroindazole, and TRIM and from juvenile (saline-treated) pigs (P < 0.01, by regression equality test); no significant difference in regression was noted among newborn animals treated with NOS inhibitors and juvenile (saline-treated) pigs (P > 0.2). Accordingly, the percent change in ChBF secondary to increasing OPP from 90 to 125 mmHg was significantly greater in newborn saline-treated pigs (44-50%) than in those treated with L-NAME, 7-NINA, 3-bromo-7-nitroindazole, and TRIM (3-10%; P < 0.01). Consequently, the changes in choroidal vascular resistance (over the same OPP range) were higher in newborns treated with L-NAME, 7-NINA, 3-bromo-7-nitroindazole, and TRIM (12-28%) than after saline (1-5%) (P < 0.01).


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  		Fig. 2.   ChBF as a function of OPP. Newborn animals (A to E) were treated intravenously with 1 mg/kg bolus followed by 50 µg · kg-1 · min-1 of one of the following agents: saline (n = 4), L-NAME (n = 3), 7-NINA (n = 3), 3-bromo-7-nitroindazole (n = 3), and 1-(2-trifluromethyl-phenyl)imidazole (TRIM; n = 3). Juvenile pigs (F) received only saline (n = 3). Each animal was subjected to stepwise acute increases in OPP from baseline to ~90, 105, and 125 mmHg; preset OPP values varied by 5 mmHg. Dotted lines correspond to the regressions for individual animals, and solid lines are mean regressions for all animals in the group. In saline-treated newborn animals, ChBF increased linearly with OPP (r = 0.78-0.91, P < 0.01). In newborn animals treated with L-NAME, 7-NINA, 3-bromo-7-nitroindazole, and TRIM and in juvenile pigs, ChBF did not change as a function of OPP (r = 0.08-0.36, P > 0.1); regression for saline-treated newborn pigs differed from all others studied (P < 0.05 by regression equality test).

Effects of hexamethonium on the upper limit of RBF and ChBF autoregulation. Because data presented so far strongly support a role for nNOS in ChBF autoregulation and because choroid NO from nNOS arises in response to autonomic (parasympathetic) nerve stimulation (34, 44), we examined whether autonomic ganglionic blockade also modulates ocular blood flow autoregulation. The ganglionic blocker hexamethonium evoked ChBF autoregulation (Fig. 3) such that ChBF did not change as a function of OPP (r = 0.15-0.29, P > 0.1). In contrast, RBF autoregulation was unaffected by hexamethonium (r = 0.78-0.91, P < 0.01); there was no significant difference in the regression of RBF as a function of OPP between saline- and hexamethonium-treated animals (P > 0.2). Data suggest autonomic nervous system control of ChBF but not of RBF autoregulation.


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  		Fig. 3.   RBF and ChBF as a function of OPP in newborn pigs. Animals were treated intravenously with saline (n = 4) or hexamethonium 1 mg/kg (n = 3). Each animal was subjected to stepwise acute increases in OPP as described in Fig. 2. Dotted lines correspond to the regressions for individual animals, and solid lines represent the mean regressions for all animals in the group. RBF and ChBF in saline-treated animals and RBF in hexamethonium-treated animals increased linearly with OPP (r = 0.68-0.91, P < 0.01). Hexamethonium prevented the change in ChBF as a function of OPP (r = 0.15-0.29, P > 0.1).

Effects of NOS inhibitors on cGMP levels in choroid. NO generation in choroid in response to increases in perfusion pressure was assessed by measurement of its major mediator cGMP (15). Basal choroidal cGMP level was higher in newborn than in juvenile animals. During increased perfusion pressure (OPP = 125 ± 5 mmHg), cGMP levels increased by nearly twofold in newborn but not in juvenile pigs (Fig. 4). Basal cGMP level was reduced by all NOS inhibitors and hexamethonium to approach values in juvenile pigs, albeit the effect of nonselective NOS inhibitor L-NAME was greater. All (nonselective and neuronal) NOS inhibitors as well as ganglionic blocker hexamethonium prevented the hypertension-induced increase in cGMP in choroid (Fig. 4).


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  		Fig. 4.   Concentrations of cGMP in choroid of pigs during normotension or subjected to increased perfusion pressure. Baseline OPP was 59 ± 5 mmHg in newborn animals and 65 ± 6 mmHg in juveniles. Saline, L-NAME, 7-NINA, 3-bromo-7-nitroindazole, or TRIM was infused intravenously for 1 h (1 mg/kg bolus followed by 50 µg · kg-1 · min-1 perfusion). Immediately after the 2-min induction of hypertension in newborn and juvenile pigs, choroids were removed for subsequent cGMP measurement. Values are means ± SE (n = 6) for each value. *P < 0.05 compared with corresponding value during normotension. dagger P < 0.01 compared with corresponding values in saline-treated newborns.

eNOS and nNOS mRNA expressions and activities in choroid. eNOS and nNOS mRNA expression and activities were greater in newborn than in juvenile choroid (Fig. 5). cNOS activity was equivalently contributed by eNOS and nNOS in both age groups; iNOS activity was minimal.


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  		Fig. 5.   Endothelial and neuronal nitric oxide synthase (eNOS and nNOS, respectively) expression and activities in choroids of newborn and juvenile pigs. A and B: total mRNA (60 µg) isolated from choroid of newborn (1-2 days old) and juvenile (4-6 wk old) pigs was subjected to RNase protection assay. C: NOS activity in choroid of newborn and juvenile pigs (see MATERIALS AND METHODS for details). Data are means ± SE of 4 separate experiments. *P < 0.05 compared with corresponding values for juveniles.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The lack of pressure-induced autoregulation of the ocular vasculature of the newborn is largely attributed to excess NO expression and activity, which mask the effects of implicated constrictors (24, 26). eNOS expression has been shown to be increased in ocular tissues of the perinate (1, 17); however, developmental profiles of nNOS expression and activity have not yet been demonstrated. The relative contribution of NO originating from eNOS and nNOS in this autoregulatory process also remains unknown. We therefore investigated whether increased NO production from nNOS during the neonatal period may contribute to curtailing RBF and ChBF autoregulation in newborn animals. Our data indicate that selective pharmacological blockade of nNOS prevents the hypertension-induced production of NO and enhances autoregulation of ChBF but not RBF of the newborn, reproducing responses in choroid of older animals. Results also support a role for the autonomic nervous system [likely parasympathetic (34, 44-46, 50)] in autoregulatory ocular blood flow response of newborn animals.

The present findings uncover a major role for increased nNOS activity in curtailing autoregulation of ChBF of the newborn. This inference is supported by a number of observations. 1) nNOS (and eNOS) expression and activity are indeed higher in newborn than in juvenile tissues (Fig. 5). Because NO production is dependent on the expression of NOS (48), increased vasorelaxation can be anticipated (27); correspondingly, newborn animals, compared with juveniles, exhibited higher nitroindazole-, TRIM- and L-NAME-inhibitable basal cGMP levels, concordant with their NOS activity (Figs. 4 and 5). 2) Acute hypertension induced a rise in cGMP levels in the newborn, and this effect was prevented by various nNOS inhibitors (Fig. 4); cGMP did not increase in juveniles that exhibit ChBF autoregulation (Fig. 2) (24, 28), possibly in part because of decreased nNOS activity (Fig. 5) as well as a greater efficacy of hypertension-induced increases in constrictors (2, 3), which may in turn reduce the microvascular shear stress and associated release of NO (42). 3) Most convincingly, distinct inhibitors of nNOS, notably the selective TRIM (22), caused ChBF to remain stable and vascular resistance to augment despite increases in OPP, unveiling a ChBF autoregulation similar to the one achieved with nonselective NOS inhibitor L-NAME, and which resembles that seen in mature animals (Figs. 1 and 2) (28).

In contrast to the choroid, nNOS inhibition did not affect RBF autoregulation of the newborn; whereas inhibition of both eNOS and nNOS using L-NAME enhanced RBF autoregulation. Because all nNOS inhibitors utilized reduced NOS activity in the central nervous system (4, 47), limited distribution of the agents to the retina is an unlikely explanation for their failure to affect RBF autoregulation. On the other hand, both retinal and choroidal circulation differ substantially in their mechanisms of regulation. The retinal vasculature, which is devoid of autonomic innervation (5), is mainly governed by local factors, whereas the choroid, which is essentially an innervated vascular tissue (14, 44, 45, 50), is particularly modulated by the autonomic nervous system (7, 44). These observations could account for the lack of effect of selective nNOS inhibitors on the upper limit of RBF in contrast to their effects on ChBF autoregulation (Figs. 1 and 2). Thus, although NO affects both RBF and ChBF autoregulation of the newborn (Fig. 1) (24), eNOS would seem to be the primary regulator in the retina and nNOS a major contributor in the choroid.

A role for the parasympathetic autonomic nervous system in autoregulation of blood flow to major organs of newborns has been reported (12, 19). The activity of this autonomic nervous system also seems to be higher in newborn than in older subjects (43). Because NO from nNOS arises largely from the autonomic parasympathetic nerve fibers in the choroid (34, 44, 50), an involvement of autonomic stimulation during autoregulatory blood flow adjustment was postulated. Our data revealed that ganglionic blockade unveiled an autoregulation of the choroid and prevented the hypertension-induced increase in cGMP as seen with nNOS inhibitors (Figs. 3 and 4), whereas hexamethonium had no effect on RBF autoregulation (Fig. 3), likely because this quaternary ammonium agent does not cross the blood-brain (retinal) barrier (36) and retinal microvessels lack peripheral autonomic innervation (5). Altogether, results suggest a NO-dependent role for the parasympathetic nervous system in impairing autoregulation of ChBF.

Although one may be tempted to suggest a predominant role for nNOS in ChBF autoregulation of the newborn, a role for eNOS cannot be fully excluded. Despite available selective eNOS inhibitors, a significant eNOS activity in the newborn (1, 17, 26), and a regulation of this isoenzyme by shear stress (42), a contribution of eNOS in ChBF autoregulation is conceivable; also, ganglionic blockade can interfere with the release of neurotransmitters, which in turn stimulate eNOS (46, 49). Furthermore, 7-nitroindazole, which unveiled a ChBF autoregulation in studies over a wide range of OPP (Fig. 1), does exhibit eNOS inhibitory activity (51). However, RBF autoregulation was unaffected by 7-nitroindazole, and other more selective nNOS inhibitors, especially TRIM (22), reproduced the same effects of 7-nitroindazole on RBF and ChBF autoregulation (Figs. 1 and 2).

In summary, data reveal that excess formation of NO from nNOS plays an important role in curtailing the upper limit of ChBF autoregulation during an acute rise in perfusion pressure in the newborn; this process seems to involve the autonomic nervous system. In contrast, RBF autoregulation of the newborn appears to be affected by eNOS. The fact that the choroid of the newborn lacks the capacity to restrict an augmentation in blood flow and, consequently, oxygen delivery to the retina (23, 26) in response to an acute increase in perfusion pressure may be relevant with regard to the pathogenesis of vasoproliferative retinopathy of the premature. One could speculate that inhibition of activity of specific NOS isoforms might be effective in the prevention of retinopathy of prematurity (11).


    ACKNOWLEDGEMENTS

We are grateful to H. Fernandez for technical assistance.


    FOOTNOTES

This work was supported by grants from the Canadian Institutes of Health Research, the Heart and Stroke Foundation of Québec, the Hospital for Sick Children Foundation, the March of Dimes Birth Defects Foundation, and the Fonds de la Recherche en Santé du Québec. P. Hardy and I. Dumont are recipients of fellowship and studentship awards, respectively, from the Canadian Institute of Health Research and the Ministry of Indian and Northern Affairs, Canada. S. Chemtob is the recipient of a Canada Research Chair (Perinatology).

Address for reprint requests and other correspondence: S. Chemtob, Depts. of Pediatrics, Ophthalmology, and Pharmacology, Research Center, Hôpital Sainte-Justine, 3175 Côte Sainte-Catherine, Montréal, Québec H3T 1C5, Canada (E-mail: sylvain.chemtob{at}umontreal.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 26 April 2001; accepted in final form 12 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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