April 2010
Volume 51, Issue 4
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Retina  |   April 2010
The Vasodilatory Effect of Juxta-arteriolar Microinjection of EndothelinA Receptor Inhibitor in Healthy and Acute Branch Retinal Vein Occlusion Minipig Retinas
Author Affiliations & Notes
  • Alexandros N. Stangos
    From the Laboratory of Neurobiology and Retinal Vascular Physiology, Ophthalmology Service, Department of Clinical Neurosciences, Geneva School of Medicine, Geneva, Switzerland; and
  • Ioannis K. Petropoulos
    From the Laboratory of Neurobiology and Retinal Vascular Physiology, Ophthalmology Service, Department of Clinical Neurosciences, Geneva School of Medicine, Geneva, Switzerland; and
  • Jean-Antoine C. Pournaras
    the Jules Gonin Eye Hospital, University of Lausanne, Lausanne, Switzerland.
  • Efstratios Mendrinos
    From the Laboratory of Neurobiology and Retinal Vascular Physiology, Ophthalmology Service, Department of Clinical Neurosciences, Geneva School of Medicine, Geneva, Switzerland; and
  • Constantin J. Pournaras
    From the Laboratory of Neurobiology and Retinal Vascular Physiology, Ophthalmology Service, Department of Clinical Neurosciences, Geneva School of Medicine, Geneva, Switzerland; and
  • Corresponding author: Constantin J. Pournaras, Ophthalmology Service, Department of Clinical Neurosciences, University Hospitals of Geneva, 22 Rue Alcide-Jentzer, 1211 Geneva, Switzerland; constantin.pournaras@hcuge.ch
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 2185-2190. doi:10.1167/iovs.09-3735
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      Alexandros N. Stangos, Ioannis K. Petropoulos, Jean-Antoine C. Pournaras, Efstratios Mendrinos, Constantin J. Pournaras; The Vasodilatory Effect of Juxta-arteriolar Microinjection of EndothelinA Receptor Inhibitor in Healthy and Acute Branch Retinal Vein Occlusion Minipig Retinas. Invest. Ophthalmol. Vis. Sci. 2010;51(4):2185-2190. doi: 10.1167/iovs.09-3735.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To investigate the effect of the endothelinA receptor inhibitor BQ-123 on the retinal arteriolar vasculature in minipig retinas in normal eyes and eyes with acute branch retinal vein occlusion (BRVO).

Methods.: Seven healthy eyes of seven minipigs and six eyes of six minipigs with experimental BRVO were evaluated under systemic anesthesia. An intravitreal juxta-arteriolar microinjection of 30 μL BQ-123 0.61 μg/mL (pH 7.4) was performed in all but one eye from each group, into which the physiologic saline vehicle alone was injected. Vessel-diameter changes were measured with a retinal vessel analyzer.

Results.: In healthy minipig retinas (n = 6), arteriolar diameter (±SD) increased 6.19% ± 3.55% (P < 0.05), 25.98% ± 2.37% (P < 0.001), 23.65% ± 1.2% (P < 0.001), and 16.84% ± 1.95% (P < 0.001), at 1, 5, 10, and 15 minutes, respectively, after BQ-123 microinjection. Two hours after experimental BRVO (n = 5), the retinal arteriolar diameter had decreased (13.07% ± 5.7%; P < 0.01). One, 5, 10, and 15 minutes after BQ-123 microinjection, retinal arteriolar diameter had increased by 7.14% ± 3.3% (P < 0.01), 26.74% ± 7.63% (P < 0.001), 23.67% ± 6.4% (P < 0.001), and 16.09% ± 3.41% (P < 0.001), respectively. Vehicle only injection had no vasoactive effect on physiologic or BRVO retinas.

Conclusions.: A significant increase in retinal arteriolar diameter was demonstrated after juxta-arteriolar BQ-123 microinjection in healthy and in acute BRVO minipig retinas. The results suggest a role for endothelin-1 in maintaining retinal basal arteriolar tone. Reversing the BRVO-related vasoconstriction by juxta-arteriolar BQ-123 microinjection could bring a new perspective to the management of BRVO.

Correctly regulated hemodynamics and delivery of oxygen and metabolic substrates, and an intact blood–retinal barrier are necessary for retinal homeostasis. Since the retina lacks sympathetic innervation and is not influenced by hormone circulation, retinal blood flow is autoregulated by the interaction of myogenic and metabolic mechanisms through the release of vasoactive substances by the vascular endothelium and retina tissue surrounding the arteriolar wall. 1 The rate of retinal blood flow is proportional to the perfusion pressure and is inversely proportional to vascular resistance, which is mainly related to retinal vessel diameter. In turn, the diameter of the retinal resistance vessels is modulated by the interaction of multiple systemic factors and local control mechanisms that affect the tone of the vascular smooth muscle cells and the capillary pericytes. 2,3  
Endothelins are a three-member peptide family of 21 amino acids, 4 secreted by a wide variety of tissues, where they act as modulators of vasomotor tone, cell proliferation, and hormone production. 5,6 Two types of endothelin receptors, endothelinA and endothelinB, with different sensibilities for the three endothelin ligands have been identified in the retina and optic nerve head in human and porcine eyes. 7 EndothelinA receptors have a high affinity for endothelin-1 and are expressed on the vascular smooth muscle and pericytes. 8,9  
Endothelin-1, is the most potent vasoconstricting factor presently known, affecting both smooth muscle cells and pericytes. 3 Increased endothelin-1 levels have been implicated in the pathogenesis of numerous ocular diseases, including glaucoma, 10 diabetic retinopathy, 11,12 retinal vein occlusion, 13,14 HIV-related retinal microangiopathic syndrome, 15 and nonarteritic anterior ischemic optic neuropathy. 16  
Research has demonstrated that systemically administered endothelinA receptor inhibitor antagonizes the powerful vasoconstrictor effect of exogenous endothelin-1 on retinal blood flow. 17 We set out to investigate the presumed vasodilatory effect of intravitreous administration of endothelinA receptor inhibitor on the minipig retinal vasculature under physiologic conditions and after experimental branch retinal vein occlusion (BRVO). 
Materials and Methods
Experiments were conducted in 13 eyes of 13 minipigs (Göttingen breed, Arare Animal Facility, Geneva, Switzerland) weighing 10 to 12 kg. The minipig's retina is an experimental model close to the human retina in both its neuroanatomic and vascular aspects. 18,19 All the experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, after permission was granted by the local veterinary authorities of Geneva. 
Animal Preparation
Minipigs were prepared for the experiments as previously described. 20 After premedication with intramuscular injection of 3 mL (15 mg) of the tranquilizer midazolanum maleate (Dormicum; Roche Pharma, Reinach, Switzerland), 3 mL (120 mg) of the tranquilizer azaperon (Stresnil; Janssen Pharmaceutica, Beerse, Belgium), and 1 mL (0.5 mg) atropine, anesthesia was induced with 2 to 3 mg ketamine hydrochloride (Ketalar; Parke-Davis, Zurich, Switzerland) injected into an ear vein. Analgesia was induced with 2 mL (100 μg) fentanyl (Sintenyl; Sintetica SA, Mendrisio, Switzerland), and curarization was performed with 2 mL (4 mg) pancuronium bromide (Pavulon; Organon SA, Pfäffikon, Switzerland). 
The animals were intubated and artificially ventilated. After arterial, venous, and bladder catheterization, anesthesia, analgesia, and myorelaxation were maintained throughout the experiment by continuous perfusion of ketamine, fentanyl, and pancuronium, respectively. 
Each animal was ventilated through a variable-volume respirator (Sulla 909 V; Dräger, Lübeck, Germany) at approximately 18 strokes/minute with a continuous flow of 20% to 25% O2 and 75% to 80% N2O. Systolic and diastolic arterial blood pressure was monitored through the femoral artery with a transducer (Minograph; Siemens-Elema, Solna, Sweden). Body temperature was maintained between 36°C and 37°C with a thermal blanket. 
Arterial partial pressure oxygen (Pao 2), carbon dioxide pressures (Paco 2), and pH were measured from the same artery with a blood gas analyzer (Labor-system; Flukiger AG, Menziken, Switzerland) and kept under control throughout the experiment by adjusting the ventilatory rate, stroke volume, and composition of the inhaled gas (i.e., O2, N2O). A head-holder was used to avoid movements from respiration. 
The upper and lower eyelids were removed, as well as a rectangular area of skin surrounding the eye; the bulbar conjunctiva was detached; the sclera was carefully cleaned up to 5 mm from the limbus; the superficial scleral vessels were thermocauterized; the globe was fixed with a metal ring sutured around the limbus; and a sclerotomy 2 to 3 mm posterior to the limbus was performed. A small contact lens with a flat exterior surface was placed on the cornea. The pupil was dilated with 1% atropine eye drops and the fundus was observed using an operating microscope (Carl Zeiss Meditec, GmbH, Oberkochen, Germany). 
Experimental Procedure
One eye of each animal was chosen for the experiment according to the best quality of fundus visualization. In all 13 eyes, a continuous (1 minute) baseline diameter recording of the preselected to study retinal arteriole was performed. In the first six eyes, after arteriole-diameter baseline recording, a juxta-arteriolar microinjection of 30 μL of endothelinA receptor inhibitor 0.61 μg/mL (BQ-123; Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany) dissolved in physiologic saline solution (PSS) was performed. Micropipettes were prepared as previously described. 21 In each case, the micropipette was introduced into the vitreous cavity through a 23-gauge pars plana sclerostomy and placed approximately 50 to 100 μm from the preselected retinal arteriole. Immediately after BQ-123 microinjection, the micropipette was removed, the sclerostomy was sealed with metal plugs, and the retina was continuously observed up to 15 minutes for changes in arteriolar diameter. 
Five eyes, after arteriole diameter baseline recording, were subjected to an experimental BRVO by a standardized photodynamic method explained in detail later. Two hours after experimental BRVO, a juxta-arteriolar microinjection of 30 μL BQ-123 0.61 μg/mL dissolved in PSS was performed on a retinal arteriole in the vicinity of the experimental BRVO site. As before, immediately after the BQ-123 microinjection, the micropipette was removed, the sclerostomy was sealed, and the retina was continuously observed up to 15 minutes, for changes in arteriole diameter. 
In our effort to investigate the effect of the vehicle or the injection itself on the diameter of the retina vasculature, we further subjected one physiologic and one BRVO minipig eye to the same procedures as just described. Instead of BQ-123, we injected the PSS vehicle alone. 
All the experiments reported herein were performed in conditions of normoxia, normocapnia, and ∼pH 7.4. Care was taken to avoid significant changes in arterial blood pressure, by adjusting the depth of anesthesia and/or the volume of intravenous fluid administration, so as to exclude any effect of blood pressure variations on the retinal arteriolar diameter. 22  
Experimental BRVO Model
A modified experimental photodynamic BRVO model, based on previous descriptions, 23,24 was introduced to simulate as accurately as possible the pathophysiological phenomena after BRVO. Accordingly, 30 mg/kg of rose bengal solution (Sigma-Aldrich GmbH) were injected via an ear vein into the systemic circulation. An endoillumination light source (GLS; Grieshaber Light Source; Grieshaber GmbH, Schaffhausen, Switzerland) was used to deliver a light beam (optic energy, 10 mW) through a fiber optic on the superotemporal or inferotemporal retinal vein, at least 0.5 disc diameters away from the optic nerve head and approximately 400 to 500 μm from the vessel subjected to BRVO. After a 10-minute period of continuous exposure to light, intravascular plaque formation with concomitant red blood cell stagnation was evident (Fig. 1A), suggesting thrombus formation. Indeed, histologic analysis of this BRVO model confirmed the formation of a massive endovascular thrombus (Fig. 1B). 
Figure 1.
 
Experimental photodynamic BRVO in the minipig. (A) Intravascular plaque formation with concomitant red blood cell stagnation (arrow), suggesting an endovascular thrombus formation. (B) Light micrograph of a retinal vein after experimental photodynamic BRVO. The lumen was occupied by a massive thrombus. Edema fluid appeared in the perivascular tissue. Stain, 1% toluidine blue.
Figure 1.
 
Experimental photodynamic BRVO in the minipig. (A) Intravascular plaque formation with concomitant red blood cell stagnation (arrow), suggesting an endovascular thrombus formation. (B) Light micrograph of a retinal vein after experimental photodynamic BRVO. The lumen was occupied by a massive thrombus. Edema fluid appeared in the perivascular tissue. Stain, 1% toluidine blue.
Retinal Arteriolar Diameter Measurements
Changes in retinal arteriolar diameter were measured with a commercially available retinal vessel analyzer (RVA; Imedos GmbH, Jena, Germany). This instrument enables a fast, noninvasive, objective evaluation of changes in retinal diameter. 2527 It comprises a fundus camera (FF 450; Carl Zeiss Meditec), a video camera, a real-time monitor, and a personal computer with vessel diameter-analysis software for the accurate determination of retinal vessel diameter. Retinal vessel diameters are analyzed in real time with a maximum frequency of 50 Hz, which means that every second a maximum of 25 readings of vessel diameter is obtained. For this purpose, the fundus is imaged onto the charge-coupled device chip of the video camera. The observed fundus images are digitalized with a frame grabber. In addition, the fundus images can be inspected on the real-time monitor and, if necessary, stored by video recorder. 
Each experiment was recorded with a fundus camera connected to a high-resolution digital video recorder. After the end of each experiment, we connected the video recorder to the RVA and performed the analysis offline from the recorded videotapes. 
Because of the absorbant properties of hemoglobin, each blood vessel has a specific transmittance profile. Measurement of retinal vessel diameters is based on adaptive algorithms that use these specific profiles. To select a region of interest, the user defines a rectangle on the screen of the real-time monitor. Thereafter, the measurement of vessel diameters can be started. Retinal vessel diameter is then calculated along the arterial segment, which lies within the rectangle. The software calculates the vessel diameter in arbitrary units (AU), which approximately correspond to micrometers at the retinal plane. The retinal vessel diameters herein are presented in AU. 
Statistical Analysis
To test the differences in retinal arteriolar diameter over the time course of the experiments, we performed repeated-measures analysis of variance (ANOVA). Post hoc comparisons were performed using paired t-tests and Bonferroni's correction for multiple comparisons. For data description, drug-induced retinal diameter changes were expressed as the percentage change from baseline. Results are presented as the mean ± SD. In all comparisons, P < 0.05 defined statistically significant differences (SPSS statistical software, ver. 15.0.0; SPSS Inc., Chicago, IL). 
Results
Measurements of mean retinal arteriolar diameter in the six healthy minipig retinas at baseline and at different time points are synopsized in Table 1. The mean retinal arteriolar diameter significantly increased after juxta-arteriolar BQ-123 microinjection, representing an increase of 6.19% ± 3.55% (P < 0.05), 25.98% ± 2.37% (P < 0.001), 23.65% ± 1.2% (P < 0.001), and 16.84% ± 1.95% (P < 0.001), at 1, 5, 10, and 15 minutes, respectively, after BQ-123 microinjection (Fig. 2). A typical experiment on a healthy minipig retina is shown in Figure 3. Retinal arteriolar diameter changes during this experiment are depicted in Figure 4
Table 1.
 
Retinal Arteriolar Diameter of Healthy Minipig Retinas
Table 1.
 
Retinal Arteriolar Diameter of Healthy Minipig Retinas
Time Course Mean Retinal Diameter
Baseline 193.22 ± 10.69
1 Minute after IVT BQ-123 205.33 ± 15.84
5 Minute after IVT BQ-123 243.39 ± 13.44
10 Minute after IVT BQ-123 238.96 ± 13.97
15 Minute after IVT BQ-123 226 ± 15.56
Figure 2.
 
Percentage change (mean ± SD) in retinal arteriolar diameter compared to baseline, measured with an RVA during the time course of the experiments on healthy minipig retinas (n = 6). Arteriolar diameter increased significantly (6.19% ± 3.55%, P < 0.05; 25.98% ± 2.37%, P < 0.001; 23.65% ± 1.2%, P < 0.001; and 16.84% ± 1.95%, P < 0.001) at 1, 5, 10, and 15 minutes after juxta-arteriolar microinjection of the endothelinA receptor inhibitor BQ-123.
Figure 2.
 
Percentage change (mean ± SD) in retinal arteriolar diameter compared to baseline, measured with an RVA during the time course of the experiments on healthy minipig retinas (n = 6). Arteriolar diameter increased significantly (6.19% ± 3.55%, P < 0.05; 25.98% ± 2.37%, P < 0.001; 23.65% ± 1.2%, P < 0.001; and 16.84% ± 1.95%, P < 0.001) at 1, 5, 10, and 15 minutes after juxta-arteriolar microinjection of the endothelinA receptor inhibitor BQ-123.
Figure 3.
 
Appearance of the healthy minipig retina. (A) Moments before juxta-arteriolar microinjection of the endothelinA receptor inhibitor BQ-123. The tip of the glass micropipette is visible (white arrow) as it approaches the site of the microinjection (white arrowhead). (B) Significant vasodilation (white arrowhead) 7 minutes after juxta-arteriolar microinjection of BQ-123.
Figure 3.
 
Appearance of the healthy minipig retina. (A) Moments before juxta-arteriolar microinjection of the endothelinA receptor inhibitor BQ-123. The tip of the glass micropipette is visible (white arrow) as it approaches the site of the microinjection (white arrowhead). (B) Significant vasodilation (white arrowhead) 7 minutes after juxta-arteriolar microinjection of BQ-123.
Figure 4.
 
Retinal arteriolar diameter changed during a typical experiment on a healthy minipig retina. Original recordings from the RVA (same experiment as in Fig. 3). The software calculates the vessel diameter in AU, which approximately correspond to micrometers at the retinal plane. Retinal arteriolar diameter markedly increased from around 180 AU at baseline to as high as 254 AU 7 minutes after juxta-arteriolar microinjection of the endothelinA receptor inhibitor BQ-123. It then slightly decreased over 15 minutes.
Figure 4.
 
Retinal arteriolar diameter changed during a typical experiment on a healthy minipig retina. Original recordings from the RVA (same experiment as in Fig. 3). The software calculates the vessel diameter in AU, which approximately correspond to micrometers at the retinal plane. Retinal arteriolar diameter markedly increased from around 180 AU at baseline to as high as 254 AU 7 minutes after juxta-arteriolar microinjection of the endothelinA receptor inhibitor BQ-123. It then slightly decreased over 15 minutes.
Measurements of mean retinal arteriolar diameter, for the five minipig retinas that were subjected to experimental BRVO, are synopsized in Table 2. For all BRVO eyes grouped together, the mean baseline retinal arteriolar diameter has significantly decreased (13.07% ± 5.77%) by 2 hours after experimental BRVO. This vasoconstriction effect, however, was reversed after juxta-arteriolar BQ-123 microinjection. Retinal arteriolar diameter increased by 7.14% ± 3.3% (P < 0.01), 26.74% ± 7.63% (P < 0.001), 23.67% ± 6.4% (P < 0.001), and 16.09% ± 3.41% (P < 0.001), 1, 5, 10, and 15 minutes after BQ-123 microinjection (Fig. 5). A typical experiment is shown in Figure 6. Retinal arteriolar diameter changes during this experiment are depicted in Figure 7
Table 2.
 
Retinal Arteriolar Diameter of Minipig Retinas after Experimental BRVO
Table 2.
 
Retinal Arteriolar Diameter of Minipig Retinas after Experimental BRVO
Time Course Mean Retinal Diameter
Baseline 193.36 ± 12.25
2 Hours after experimental BRVO 167.95 ± 13.52
1 Minute after IVT BQ-123 179.93 ± 15.07
5 Minute after IVT BQ-123 212.09 ± 6.5
10 Minute after IVT BQ-123 207.02 ± 5.46
15 Minute after IVT BQ-123 194.74 ± 12.70
Figure 5.
 
Percentage change (mean ± SD) in retinal arteriolar diameter compared with baseline, measured with an RVA during the time course of the experiments (n = 5). Two hours after experimental BRVO, arteriolar diameter had significantly decreased (13.07% ± 5.7%; P < 0.01). Arteriolar diameter increased significantly (7.14% ± 3.3%; P < 0.01; 26.74% ± 7.63%, P < 0.001; 23.67% ± 6.4%, P < 0.001; and 16.09% ± 3.41%, P < 0.001) at 1, 5, 10, and 15 minutes after juxta-arteriolar microinjection of the endothelinA receptor inhibitor BQ-123.
Figure 5.
 
Percentage change (mean ± SD) in retinal arteriolar diameter compared with baseline, measured with an RVA during the time course of the experiments (n = 5). Two hours after experimental BRVO, arteriolar diameter had significantly decreased (13.07% ± 5.7%; P < 0.01). Arteriolar diameter increased significantly (7.14% ± 3.3%; P < 0.01; 26.74% ± 7.63%, P < 0.001; 23.67% ± 6.4%, P < 0.001; and 16.09% ± 3.41%, P < 0.001) at 1, 5, 10, and 15 minutes after juxta-arteriolar microinjection of the endothelinA receptor inhibitor BQ-123.
Figure 6.
 
Appearance of the minipig retina after experimental photodynamic BRVO. (A) Fundus photograph 2 hours after BRVO and moments before juxta-arteriolar microinjection (white arrowhead) of the endothelinA receptor inhibitor BQ-123. (B) Significant vasodilatation (white arrowhead) 6 minutes after juxta-arteriolar microinjection of BQ-123.
Figure 6.
 
Appearance of the minipig retina after experimental photodynamic BRVO. (A) Fundus photograph 2 hours after BRVO and moments before juxta-arteriolar microinjection (white arrowhead) of the endothelinA receptor inhibitor BQ-123. (B) Significant vasodilatation (white arrowhead) 6 minutes after juxta-arteriolar microinjection of BQ-123.
Figure 7.
 
Retinal arteriolar diameter changed during a typical experiment on a minipig retina after experimental BRVO. Original recordings from the RVA (same experiment as in Fig. 6). The software calculates the vessel diameter in AU, which approximately correspond to micrometers at the retinal plane. Retinal arteriolar diameter markedly decreased from 190 AU at baseline to 157 AU 2 hours after experimental BRVO. Diameter increased to 211 AU 6 minutes after juxta-arteriolar microinjection of the endothelinA receptor inhibitor BQ-12 and then decreased over 15 minutes.
Figure 7.
 
Retinal arteriolar diameter changed during a typical experiment on a minipig retina after experimental BRVO. Original recordings from the RVA (same experiment as in Fig. 6). The software calculates the vessel diameter in AU, which approximately correspond to micrometers at the retinal plane. Retinal arteriolar diameter markedly decreased from 190 AU at baseline to 157 AU 2 hours after experimental BRVO. Diameter increased to 211 AU 6 minutes after juxta-arteriolar microinjection of the endothelinA receptor inhibitor BQ-12 and then decreased over 15 minutes.
The vasodilatory effect after BQ-123 juxta-arteriolar microinjection was transient. Both in healthy and in BRVO minipig retinas, vasodilatation reached its maximum at 5 to 7 minutes after the microinjection and then decreased over 15 minutes. Vehicle-only microinjection (PSS) had no effect on either normal or BRVO-affected retinas. 
Discussion
Endothelins are a three-member peptide family of 21 amino acids 4 that act as modulators of vasomotor tone, cell proliferation, and hormone production. 5,6 These peptides are important in vascular physiology and disease. Endothelin-1, the most potent vasoconstrictive substance known, is produced by vascular smooth-muscle cells, central nervous system neurons, and astrocytes, and it is the only endothelin family member produced in endothelial cells. The action of endothelin-1 is mediated mainly by the activation of endothelinA receptors. 28 These receptors, present on vascular smooth muscle cells and capillary pericytes, 8,9 are characterized by a very high affinity for endothelin-1. The combination of the local paracrine effects of endothelin-1, alongside nitric oxide and other vasoactive factor release, play a relevant physiological role in the regulation of retinal blood flow, 1 especially because of the absence of autonomic innervation on retinal vessels. 
Our results demonstrate a significant increase in retinal arteriolar diameter after juxta-arteriolar endothelinA receptor inhibitor microinjection, both in healthy and in acute BRVO minipig retinas. Five minutes after BQ-123 microinjection, arteriolar diameter increased by 25.98% ± 2.37% (P < 0.001) and 26.74% ± 7.63% (P < 0.001) in healthy and BRVO retinas, respectively. It had been postulated that endothelin-1 does not play a major role in the maintenance of retinal basal vascular tone. 17 The results of the present study, however, show that BQ-123 can influence retinal hemodynamic parameters when locally administered in healthy minipig retinas. This finding suggests that endothelin-1 plays an important role in maintaining retinal basal vascular tone. 
Other experimental studies have pointed out the important role of endothelin-1 in retinal vascular disease. Endothelin-1 has been implicated in retinal vascular abnormalities of diabetic rats, 29 whereas intravitreal endothelin-1 injection resulted in retinal capillary nonperfusion and closure of retinal arterioles, 3032 implicating endothelin-1 in retinal ischemia. 
Retinal vein occlusion entails the presence of endothelial damage with venous stasis and ongoing enhanced thrombin release, conditions that, alongside hypoxia, have been extensively documented to stimulate endothelin-1 production. 3339 Previous studies have demonstrated that the development of extended areas of nonperfused capillaries after BRVO in humans correlates with the secondary constriction of the arteriole that crosses the occluded area. 40 Arteriolar constriction of the apparently nonaffected arterioles irrigating the territory of the occluded veins occurs within hours of the occlusive phenomenon and can persist for months. 41 Furthermore, the vasoconstrictive properties of endothelin-1 are greatly heightened in atherosclerotic vessels in which the opposing biological effect of nitric oxide is blunted. 42 Indeed, the sustained arteriolar vasoconstriction is strongly related to a decrease in production and release of nitric oxide by the retina. 43 Local administration of nitric oxide donors showed evidence of reversing the arteriolar vasoconstriction observed after experimental BRVO. 44  
Elevated endothelin-1 levels are a well-known marker of vascular endothelial dysfunction. 4547 Iannaccone et al. 13 have shown that patients with retinal vein occlusion have increased circulating levels of endothelin-1. They concluded that the elevated levels of endothelin-1 are the result of the occlusive event, representing a marker of the occlusion, possibly as a “spillover” phenomenon from a locally activated system. They also suggest that a self-reinforcing circle could develop locally, in which endothelin-1 released by endothelial cells after retinal vein occlusion could diffuse in the vicinity of the occluded vessel to the abluminal side of neighboring pericytes and induce capillary nonperfusion. The ensuing ischemia could further enhance endothelin-1 release by retinal endothelial cells, maintaining and extending retinal nonperfusion. 48  
Since endothelin-1 action is mediated mainly by the activation of endothelinA receptors, 28 administration of endothelinA receptor antagonists could counteract the vasoconstrictive effects in retinal vasculature exerted by endothelin-1 after BRVO. In perfused porcine eye, the introduction of endothelinA receptor antagonist inhibited endothelin-1-induced vasoconstriction and decreased flow. 49 Infusion of the selective endothelinA receptor antagonist BQ-123 into the brachial artery of normal subjects induced a 64% increase in forearm blood flow. 50 However, when systemically administered, BQ-123 had no effect on retinal hemodynamic parameters. 17 It is possible that exogenous BQ-123 cannot cross the blood–retina barrier. 51 Blood flow autoregulation is defined as the capability of an organ to regulate its blood supply in accordance with its needs. 52 This capability implies that blood flow is actively regulated by metabolism through the action of local factors that modulate the tone of the retinal resistant vessels. These factors are released by the vascular endothelium and/or neuronal tissue surrounding the vessels. Thus, local administration of vasoactive substances such as BQ-123 would be expected to exert a greater effect on tissues. 
Our results demonstrate that when locally administered, endothelinA receptor inhibitor effectively reversed the vasoconstriction that occurs within hours after experimental BRVO. In both normal and BRVO retinas, BQ-123 microinjection induced similar percentages of vasodilatation. However, the absolute values show that the arteriolar diameter after BQ-123 microinjection in healthy retinas was greater than the arteriolar diameter in BRVO retinas, probably caused by the contribution of nitric oxide to the maintenance of retinal arteriolar diameter, which is decreased in BRVO retinas 43 and is not restored after BQ-123 microinjection. This study was not specifically designed to investigate the role of endothelin-1 in retinal arteriolar vasoconstriction secondary to BRVO, and since there was no difference in the vasodilation after BQ-123 microinjection between healthy and BRVO retinas, we cannot make definite conclusions on the role of endothelin-1 in BRVO. 
In conclusion, the results of the present study provided evidence of the role of endothelin-1 in maintaining retinal basal vascular tone. In contrast to systemic administration, intravitreous administration of an endothelinA receptor antagonist appears to be a potential approach to counterbalancing the ischemic effects that occur after retinal vein occlusion, improving the supply of oxygen and nutrients to the injured tissue. The present study cast light on retinal blood flow regulation by indicating that direct effects on the retina dominate, whereas it opens new perspectives in the management of retinal vein occlusive diseases such as BRVO. 
Footnotes
 Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2007.
Footnotes
 Supported by Swiss National Science Foundation Grant 3200B0-105809.
Footnotes
 Disclosure: A.N. Stangos, None; I.K. Petropoulos, None; J.-A.C. Pournaras, None; E. Mendrinos, None; C.J. Pournaras, None
The authors thank Nicole Gilodi for technical assistance during preparation and conduct of animal experiments. 
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Figure 1.
 
Experimental photodynamic BRVO in the minipig. (A) Intravascular plaque formation with concomitant red blood cell stagnation (arrow), suggesting an endovascular thrombus formation. (B) Light micrograph of a retinal vein after experimental photodynamic BRVO. The lumen was occupied by a massive thrombus. Edema fluid appeared in the perivascular tissue. Stain, 1% toluidine blue.
Figure 1.
 
Experimental photodynamic BRVO in the minipig. (A) Intravascular plaque formation with concomitant red blood cell stagnation (arrow), suggesting an endovascular thrombus formation. (B) Light micrograph of a retinal vein after experimental photodynamic BRVO. The lumen was occupied by a massive thrombus. Edema fluid appeared in the perivascular tissue. Stain, 1% toluidine blue.
Figure 2.
 
Percentage change (mean ± SD) in retinal arteriolar diameter compared to baseline, measured with an RVA during the time course of the experiments on healthy minipig retinas (n = 6). Arteriolar diameter increased significantly (6.19% ± 3.55%, P < 0.05; 25.98% ± 2.37%, P < 0.001; 23.65% ± 1.2%, P < 0.001; and 16.84% ± 1.95%, P < 0.001) at 1, 5, 10, and 15 minutes after juxta-arteriolar microinjection of the endothelinA receptor inhibitor BQ-123.
Figure 2.
 
Percentage change (mean ± SD) in retinal arteriolar diameter compared to baseline, measured with an RVA during the time course of the experiments on healthy minipig retinas (n = 6). Arteriolar diameter increased significantly (6.19% ± 3.55%, P < 0.05; 25.98% ± 2.37%, P < 0.001; 23.65% ± 1.2%, P < 0.001; and 16.84% ± 1.95%, P < 0.001) at 1, 5, 10, and 15 minutes after juxta-arteriolar microinjection of the endothelinA receptor inhibitor BQ-123.
Figure 3.
 
Appearance of the healthy minipig retina. (A) Moments before juxta-arteriolar microinjection of the endothelinA receptor inhibitor BQ-123. The tip of the glass micropipette is visible (white arrow) as it approaches the site of the microinjection (white arrowhead). (B) Significant vasodilation (white arrowhead) 7 minutes after juxta-arteriolar microinjection of BQ-123.
Figure 3.
 
Appearance of the healthy minipig retina. (A) Moments before juxta-arteriolar microinjection of the endothelinA receptor inhibitor BQ-123. The tip of the glass micropipette is visible (white arrow) as it approaches the site of the microinjection (white arrowhead). (B) Significant vasodilation (white arrowhead) 7 minutes after juxta-arteriolar microinjection of BQ-123.
Figure 4.
 
Retinal arteriolar diameter changed during a typical experiment on a healthy minipig retina. Original recordings from the RVA (same experiment as in Fig. 3). The software calculates the vessel diameter in AU, which approximately correspond to micrometers at the retinal plane. Retinal arteriolar diameter markedly increased from around 180 AU at baseline to as high as 254 AU 7 minutes after juxta-arteriolar microinjection of the endothelinA receptor inhibitor BQ-123. It then slightly decreased over 15 minutes.
Figure 4.
 
Retinal arteriolar diameter changed during a typical experiment on a healthy minipig retina. Original recordings from the RVA (same experiment as in Fig. 3). The software calculates the vessel diameter in AU, which approximately correspond to micrometers at the retinal plane. Retinal arteriolar diameter markedly increased from around 180 AU at baseline to as high as 254 AU 7 minutes after juxta-arteriolar microinjection of the endothelinA receptor inhibitor BQ-123. It then slightly decreased over 15 minutes.
Figure 5.
 
Percentage change (mean ± SD) in retinal arteriolar diameter compared with baseline, measured with an RVA during the time course of the experiments (n = 5). Two hours after experimental BRVO, arteriolar diameter had significantly decreased (13.07% ± 5.7%; P < 0.01). Arteriolar diameter increased significantly (7.14% ± 3.3%; P < 0.01; 26.74% ± 7.63%, P < 0.001; 23.67% ± 6.4%, P < 0.001; and 16.09% ± 3.41%, P < 0.001) at 1, 5, 10, and 15 minutes after juxta-arteriolar microinjection of the endothelinA receptor inhibitor BQ-123.
Figure 5.
 
Percentage change (mean ± SD) in retinal arteriolar diameter compared with baseline, measured with an RVA during the time course of the experiments (n = 5). Two hours after experimental BRVO, arteriolar diameter had significantly decreased (13.07% ± 5.7%; P < 0.01). Arteriolar diameter increased significantly (7.14% ± 3.3%; P < 0.01; 26.74% ± 7.63%, P < 0.001; 23.67% ± 6.4%, P < 0.001; and 16.09% ± 3.41%, P < 0.001) at 1, 5, 10, and 15 minutes after juxta-arteriolar microinjection of the endothelinA receptor inhibitor BQ-123.
Figure 6.
 
Appearance of the minipig retina after experimental photodynamic BRVO. (A) Fundus photograph 2 hours after BRVO and moments before juxta-arteriolar microinjection (white arrowhead) of the endothelinA receptor inhibitor BQ-123. (B) Significant vasodilatation (white arrowhead) 6 minutes after juxta-arteriolar microinjection of BQ-123.
Figure 6.
 
Appearance of the minipig retina after experimental photodynamic BRVO. (A) Fundus photograph 2 hours after BRVO and moments before juxta-arteriolar microinjection (white arrowhead) of the endothelinA receptor inhibitor BQ-123. (B) Significant vasodilatation (white arrowhead) 6 minutes after juxta-arteriolar microinjection of BQ-123.
Figure 7.
 
Retinal arteriolar diameter changed during a typical experiment on a minipig retina after experimental BRVO. Original recordings from the RVA (same experiment as in Fig. 6). The software calculates the vessel diameter in AU, which approximately correspond to micrometers at the retinal plane. Retinal arteriolar diameter markedly decreased from 190 AU at baseline to 157 AU 2 hours after experimental BRVO. Diameter increased to 211 AU 6 minutes after juxta-arteriolar microinjection of the endothelinA receptor inhibitor BQ-12 and then decreased over 15 minutes.
Figure 7.
 
Retinal arteriolar diameter changed during a typical experiment on a minipig retina after experimental BRVO. Original recordings from the RVA (same experiment as in Fig. 6). The software calculates the vessel diameter in AU, which approximately correspond to micrometers at the retinal plane. Retinal arteriolar diameter markedly decreased from 190 AU at baseline to 157 AU 2 hours after experimental BRVO. Diameter increased to 211 AU 6 minutes after juxta-arteriolar microinjection of the endothelinA receptor inhibitor BQ-12 and then decreased over 15 minutes.
Table 1.
 
Retinal Arteriolar Diameter of Healthy Minipig Retinas
Table 1.
 
Retinal Arteriolar Diameter of Healthy Minipig Retinas
Time Course Mean Retinal Diameter
Baseline 193.22 ± 10.69
1 Minute after IVT BQ-123 205.33 ± 15.84
5 Minute after IVT BQ-123 243.39 ± 13.44
10 Minute after IVT BQ-123 238.96 ± 13.97
15 Minute after IVT BQ-123 226 ± 15.56
Table 2.
 
Retinal Arteriolar Diameter of Minipig Retinas after Experimental BRVO
Table 2.
 
Retinal Arteriolar Diameter of Minipig Retinas after Experimental BRVO
Time Course Mean Retinal Diameter
Baseline 193.36 ± 12.25
2 Hours after experimental BRVO 167.95 ± 13.52
1 Minute after IVT BQ-123 179.93 ± 15.07
5 Minute after IVT BQ-123 212.09 ± 6.5
10 Minute after IVT BQ-123 207.02 ± 5.46
15 Minute after IVT BQ-123 194.74 ± 12.70
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