May 2011
Volume 52, Issue 6
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Retina  |   May 2011
Vasomotor Effect of Intravitreal Juxta-arteriolar Injection of l-Lactate on the Retinal Arterioles after Acute Branch Retinal Vein Occlusion in Minipigs
Author Affiliations & Notes
  • Efstratios Mendrinos
    From the Laboratory of Neurobiology and Physiology of the Retinal Circulation, Department of Ophthalmology, Geneva University Hospitals, Geneva, Switzerland; and
  • Ioannis K. Petropoulos
    From the Laboratory of Neurobiology and Physiology of the Retinal Circulation, Department of Ophthalmology, Geneva University Hospitals, Geneva, Switzerland; and
  • Georgios Mangioris
    From the Laboratory of Neurobiology and Physiology of the Retinal Circulation, Department of Ophthalmology, Geneva University Hospitals, Geneva, Switzerland; and
  • Miltiadis K. Tsilimbaris
    the Department of Ophthalmology, University of Heraklion, Crete, Greece.
  • Domniki N. Papadopoulou
    From the Laboratory of Neurobiology and Physiology of the Retinal Circulation, Department of Ophthalmology, Geneva University Hospitals, Geneva, Switzerland; and
  • Aliki Geka
    From the Laboratory of Neurobiology and Physiology of the Retinal Circulation, Department of Ophthalmology, Geneva University Hospitals, Geneva, Switzerland; and
  • Constantin J. Pournaras
    From the Laboratory of Neurobiology and Physiology of the Retinal Circulation, Department of Ophthalmology, Geneva University Hospitals, Geneva, Switzerland; and
  • Corresponding author: Constantin J. Pournaras, Vitreoretinal Unit, Department of Ophthalmology, University Hospitals of Geneva, 22 Alcide-Jentzer Street, CH-1211 Geneva 14, Switzerland; constantin.pournaras@hcuge.ch
Investigative Ophthalmology & Visual Science May 2011, Vol.52, 3215-3220. doi:10.1167/iovs.10-6888
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      Efstratios Mendrinos, Ioannis K. Petropoulos, Georgios Mangioris, Miltiadis K. Tsilimbaris, Domniki N. Papadopoulou, Aliki Geka, Constantin J. Pournaras; Vasomotor Effect of Intravitreal Juxta-arteriolar Injection of l-Lactate on the Retinal Arterioles after Acute Branch Retinal Vein Occlusion in Minipigs. Invest. Ophthalmol. Vis. Sci. 2011;52(6):3215-3220. doi: 10.1167/iovs.10-6888.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose.: To investigate the effect of l-lactate on retinal arteriolar diameter after acute branch retinal vein occlusion (BRVO) in minipigs.

Methods.: Thirteen eyes of 13 minipigs were evaluated, with the animals under general anesthesia. BRVO was induced by a standard method of argon laser endophotocoagulation. Two hours after BRVO, an intravitreal, juxta-arteriolar microinjection of 50 μL l-lactate 0.5 M (pH 7.4) was performed in nine eyes. Four eyes received a microinjection of 50 μL of the solvent (pH 7.4) that was used to prepare the solution of l-lactate and served as controls. Retinal arteriolar diameter changes were measured using a retinal vessel analyzer.

Results.: Overall (n = 13), 2 hours after BRVO, there was a 9.0% ± 1.4% decrease in the retinal arteriolar diameter in the affected ares compared to baseline (P < 0.001). An increase of 26.2% ± 8.2% (P = 0.004) of the arteriolar diameter was evidenced 5 minutes after l-lactate juxta-arteriolar microinjection (n = 9) compared with the diameter before l-lactate microinjection. Thereafter, the vasodilatory effect of l-lactate persisted and remained significant until the end of the study period (27.7% ± 7.8% at 30 minutes) compared with the diameter before l-lactate microinjection (P = 0.002). Microinjection of the solvent alone (n = 4) did not produce any significant effect on the retinal arterioles, which remained constricted at all time-points (P > 0.1).

Conclusions.: These findings demonstrate a significant arteriolar vasodilation after intravitreal juxta-arteriolar l-lactate microinjection in eyes with experimental BRVO in the affected areas. l-lactate microinjection can reverse the arteriolar vasoconstriction that occurs in acute experimental BRVO.

Lactate is the major metabolite produced by anaerobic glycolysis in most tissues, but it is also produced under aerobic conditions in the retina. 1 Animal and human studies have shown that lactate increases retinal blood flow, 2 4 induces dilation of retinal arteries 5 7 and therefore is an important mediator of the metabolic retinal blood flow autoregulation. 8 Nitric oxide (NO) has been suggested as the major mediator that causes lactate-dependent vasodilation in isolated porcine retinal arterioles. 6 In vivo, neuronal/glial-derived NO may play a more important role than endothelium-derived NO in mediating the vasoactivity of l-lactate. 9  
After branch retinal vein occlusion (BRVO), the retinal territory affected by the occlusion becomes hypoxic, 10 12 and the arteriole crossing the occluded ares is often constricted. 13 Retinal arteriolar vasoconstriction corresponds with a decrease in preretinal NO concentration ([NO]) and contributes to the development of inner retinal hypoxia. 14 This vasoconstriction correlates with the development of extended ares of nonperfused capillaries and is reversed by local administration of the NO donor sodium nitroprusside, 15 suggesting that decreased NO generation is responsible for arteriolar nonperfusion after BRVO. Εndothelin-1 is another potent mediator of the retinal arteriolar vasoconstriction that occurs in BRVO. 16  
Recently, we showed that injection of intravitreal (IVT) l-arginine, the substrate from which the ubiquitous mediator NO is synthesized, can reverse the retinal arteriolar vasoconstriction that occurs after BRVO. 17 The purpose of the present study was to investigate the effect of IVT juxta-arteriolar microinjection of l-lactate on the retinal arteriolar diameter after acute BRVO in minipigs. We used a retinal vessel analyzer (RVA) which is accurate enough to detect subtle changes in vessel diameter. 18,19 Retinal vessel diameter is a useful surrogate for retinal perfusion, with changes in the diameter of the retinal arterioles, indicating changes in retinal capillary blood flow. 20  
Our working hypothesis was that IVT l-lactate, given its mechanism of action, can also reverse the retinal arteriolar vasoconstriction that occurs after BRVO, thus leading to an increase in the retinal arteriolar blood flow and possibly to improvement of the inner retinal oxygenation and cellular function in the occluded area. 
Materials and Methods
Experiments were conducted in one eye of 13 minipigs (Göttingen breed; Arare Animal Facility, Geneva, Switzerland) weighing 10 to 12 kg. Minipig retina is an experimental model close to the human retina in both the neuroanatomic and the vascular aspects. 21,22 All the experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Animal Preparation
The minipigs were prepared for the experiments as previously described. 23 After premedication with intramuscular injection of 3 mL (15 mg) of the tranquilizer midazolam maleate (Dormicum; Roche Pharma, Reinach, Switzerland), 3 mL (120 mg) of the tranquilizer azaperone (Stresnil; Janssen Pharmaceutica, Beerse, Belgium), and 1 mL (0.5 mg) atropine, anesthesia was induced with 2 to 3 mg of 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 at approximately 18 strokes/min, with a continuous flow of 20% O2 and 80% N2O, through a variable-volume respirator (Sulla 909 V; Drägerwerk AG, Liibeck, Germany). 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 pressure (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 ventilatory rate, stroke volume, and composition of the inhaled gas. 
A head holder was used to avoid movements from respiration. 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 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
With the animals under general anesthesia, 13 eyes of 13 minipigs were evaluated. BRVO was performed in all eyes by a standard method of argon laser endophotocoagulation. The first branch of one of the main veins emerging from the optic nerve head was photocoagulated, with the argon green wavelength (514 nm). Venous occlusion was achieved with a power of 250 mW, pulse duration of 0.5 seconds, and a spot diameter of ∼500 μm. The eyes were not vitrectomized. Two sclerotomies were performed using the one-step 20-gauge sutureless transconjunctival system with trocars (20-gauge; Synergetics France SARL, Paris, France). The fiber optic was inserted through the one sclerotomy and was kept stable with a micromanipulator at the same distance from the retina though the whole experiment, and the second sclerotomy was used for the IVT injection. During the experiment the opening of the second trocar was kept sealed by its plug so that no leakage was possible. 
Two hours after BRVO, an intravitreal juxta-arteriolar microinjection of 50 μL l-lactate (purchased from Sigma-Aldrich Chemie Gmbh, Deisenhofen, Germany) 0.5 M (pH 7.4) was performed in nine eyes. Four eyes received a microinjection of 50 μL of the solvent (physiologic saline solution, PSS; pH 7.4) that was used to prepare the solution of l-lactate, and these eyes served as the control. 
Microinjections were performed through a micropipette. Micropipettes (tip diameter, ∼50 μm) were pulled from borosilicate capillaries. The micropipette was introduced into the vitreous cavity through the pars plana sclerotomy and placed at a distance of 50 to 100 μm from a retinal arteriole. When injection was performed (50 μL) through the second trocar, any possible transient IOP increase was probably counterbalanced, as this trocar was left unplugged for some seconds. 
All the experiments reported herein were performed in conditions of normoxia, normocapnia, and pH 7.4 (Table 1). Care was taken to avoid significant changes in arterial blood pressure by adjusting accordingly the dosage and administration rate of the anesthetics, so as to exclude any effect of blood pressure variations on the retinal arteriolar diameter. 24 The systolic (BPsys) and diastolic (BPdia) arterial blood pressure was measured before each RVA measurement. The mean arterial pressure (MAP) was calculated according to the equation: MAP = BPdia + ⅓(BPsys − BPdia) (Table 1). Intraocular pressure was not monitored. 
Table 1.
 
Retinal Arteriolar Diameter after IVT Juxta-arteriolar Microinjection of l-lactate in Experimental BRVO
Table 1.
 
Retinal Arteriolar Diameter after IVT Juxta-arteriolar Microinjection of l-lactate in Experimental BRVO
Time Course Retinal Diameter (AU) MAP pH Po 2 Pco 2
Baseline 210 ± 48 88.0 ± 4.4 7.38 ± 0.01 105.0 ± 1.6 39.2 ± 0.5
BRVO, 2h 195 ± 43 87.1 ± 4.5 7.38 ± 0.01 107.1 ± 1.7 39.1 ± 0.4
IVT l-lactate, 5 min 241 ± 48 86.1 ± 4.5 7.38 ± 0.02 107.4 ± 1.8 38.7 ± 0.6
IVT l-lactate, 10 min 242 ± 48 85.0 ± 4.6 7.38 ± 0.01 108.0 ± 1.7 39.7 ± 0.9
IVT l-lactate, 15 min 244 ± 45 85.6 ± 4.5 7.39 ± 0.01 107.9 ± 2.3 37.6 ± 0.3
IVT l-lactate, 20 min 243 ± 44 85.5 ± 4.5 7.39 ± 0.02 108.1 ± 2.3 38.1 ± 0.3
IVT l-lactate, 25 min 244 ± 43 85.4 ± 4.5 7.39 ± 0.02 107.5 ± 2.6 37.9 ± 0.6
IVT l-lactate, 30 min 243 ± 44 85.9 ± 4.5 7.39 ± 0.02 108.6 ± 2.2 37.8 ± 0.5
Retinal Arteriolar Diameter Measurements
Retinal arteriolar diameter changes were measured with a commercially available retinal vessel analyzer (RVA; Carl Zeiss Meditec, Jena, Germany). This instrument enables a fast, noninvasive, and objective evaluation of changes in retinal diameter. 19,25 It comprises a fundus camera (FF 450; Carl Zeiss Meditec), a video camera, a real-time monitor, and a personal computer with vessel diameter-analyzing software for the accurate determination of retinal vessel diameter. 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 using a video recorder. We recorded each experimentation using a fundus camera integrated within the microscope connected to a high-resolution digital video recorder. After the end of each experimentation, we connected the video recorder to the RVA and performed the analysis off-line from the recorded digital video-tapes, using the RVA software. The reproducibility and repeatability of the RVA measurements in our animal experimental model was previously determined. 26  
Because of the absorbing properties of hemoglobin each blood vessel has a specific transmittance profile. Measurement of retinal vessel diameters is based on adaptive algorithms using 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. To study of the effect of lactate and PSS on retinal arteriolar diameter, average values of the diameter of the study arteriolar segment over 60 seconds at each time-point were considered. 
Statistical Analysis
Data were tested for normal distribution using the Kolmogorov-Smirnov test. To test differences in retinal arteriolar diameter over the time course of the experiments, the Wilcoxon signed rank test was performed when compared data did not show normal distribution, and paired t-test was performed when compared data showed normal distribution. For data description, drug-induced retinal diameter changes were expressed as the percentage change from baseline. Results are presented as the mean ± SEM. The Pearson correlation was used to evaluate whether l-lactate-induced retinal arteriolar dilation correlated with the arteriolar constriction 2 hours after BRVO. In all comparisons, P < 0.05 defined statistically significant differences. 
Results
Two hours after BRVO (n = 13) there was a 9.0% ± 1.4% decrease in the retinal arteriolar diameter in the affected areas compared with baseline (P < 0.001). An increase of 26.2% ± 8.2% (P = 0.004) of the arteriolar diameter was evidenced 5 minutes after l-lactate juxta-arteriolar microinjection (n = 9), compared with the diameter before l-lactate microinjection. Thereafter, the vasodilatory effect of l-lactate persisted and remained significant until the end of the study period, compared with the diameter before l-lactate microinjection (26.8% ± 7.8%, P = 0.003; 27.9% ± 7.7%, P = 0.002; 27.5% ± 7.6%, P = 0.002; 27.9% ± 7.6%, P = 0.002; and 27.7% ± 7.8%, P = 0.002 at 10, 15, 20, 25, and 30 minutes, respectively). We found no correlation between BRVO-induced arteriolar constriction at 2 hours and l-lactate-induced arteriolar dilation at 30 minutes (r = −0.2, P = 0.6). Retinal arteriolar diameters are presented in Table 1 in AU. Figure 1 illustrates the percentage change (mean ± SEM) of retinal diameter after juxta-arteriolar injection of l-lactate. A typical experiment is shown in Figure 2
Figure 1.
 
Percentage change (mean ± SEM) of the retinal arteriolar diameter, measured with an RVA, after juxta-arteriolar l-lactate microinjection in experimental BRVO (n = 9). Two hours after BRVO, retinal arteriolar diameter decreased by 7.2% ± 3.3% below baseline (P = 0.004). An increase of 26.2% ± 8.2% (P = 0.004) in arteriolar diameter was evidenced 5 minutes after l-lactate juxta-arteriolar microinjection (n = 9) compared with the diameter before l-lactate microinjection. Thereafter, the vasodilatory effect of l-lactate persisted and remained significant until the end of the study period compared with the diameter before l-lactate microinjection. (26.8% ± 7.8%, P = 0.003; 27.9% ± 7.7%, P = 0.002; 27.5% ± 7.6%, P = 0.002; and 27.9% ± 7.6%, P = 0.002; and 27.7% ± 7.8%, P = 0.002 at 10, 15, 20, 25 and 30 minutes, respectively).
Figure 1.
 
Percentage change (mean ± SEM) of the retinal arteriolar diameter, measured with an RVA, after juxta-arteriolar l-lactate microinjection in experimental BRVO (n = 9). Two hours after BRVO, retinal arteriolar diameter decreased by 7.2% ± 3.3% below baseline (P = 0.004). An increase of 26.2% ± 8.2% (P = 0.004) in arteriolar diameter was evidenced 5 minutes after l-lactate juxta-arteriolar microinjection (n = 9) compared with the diameter before l-lactate microinjection. Thereafter, the vasodilatory effect of l-lactate persisted and remained significant until the end of the study period compared with the diameter before l-lactate microinjection. (26.8% ± 7.8%, P = 0.003; 27.9% ± 7.7%, P = 0.002; 27.5% ± 7.6%, P = 0.002; and 27.9% ± 7.6%, P = 0.002; and 27.7% ± 7.8%, P = 0.002 at 10, 15, 20, 25 and 30 minutes, respectively).
Figure 2.
 
Retinal arteriolar diameter changes during a typical experiment in a minipig eye. (A) Fundus photograph at baseline showing the study retinal arteriole in this experiment. (B) Site of laser photocoagulation which was performed on the retinal vein. Two hours after BRVO, there was vasoconstriction of the retinal arteriole compared with baseline, measured with the RVA. (C) Thirty minutes after a juxta-arteriolar microinjection of 50 μL l-lactate 0.5 M (pH 7.4), there was a significant vasodilation of the retinal arteriole.
Figure 2.
 
Retinal arteriolar diameter changes during a typical experiment in a minipig eye. (A) Fundus photograph at baseline showing the study retinal arteriole in this experiment. (B) Site of laser photocoagulation which was performed on the retinal vein. Two hours after BRVO, there was vasoconstriction of the retinal arteriole compared with baseline, measured with the RVA. (C) Thirty minutes after a juxta-arteriolar microinjection of 50 μL l-lactate 0.5 M (pH 7.4), there was a significant vasodilation of the retinal arteriole.
Microinjection of the solvent that was used to prepare the solution of l-lactate (n = 4) did not produce any significant effect on the diameter of the retinal arterioles, which remained constricted at all time points (P > 0.1; Fig. 3). 
Figure 3.
 
Percentage change (mean ± SEM) of the retinal arteriolar diameter, measured with the RVA after juxta-arteriolar microinjection of the solvent (PSS) used for the preparation of l-lactate solution in experimental BRVO (n = 4). Two hours after BRVO, retinal arteriolar diameter decreased by 13% ± 3% below baseline (P = 0.026). Microinjection of 50 μL PSS (pH 7.4) did not produce any significant effect on the retinal arterioles, which remained constricted at all time-points (P > 0.1).
Figure 3.
 
Percentage change (mean ± SEM) of the retinal arteriolar diameter, measured with the RVA after juxta-arteriolar microinjection of the solvent (PSS) used for the preparation of l-lactate solution in experimental BRVO (n = 4). Two hours after BRVO, retinal arteriolar diameter decreased by 13% ± 3% below baseline (P = 0.026). Microinjection of 50 μL PSS (pH 7.4) did not produce any significant effect on the retinal arterioles, which remained constricted at all time-points (P > 0.1).
Discussion
In the present study, we found that juxta-arteriolar administration of l-lactate induced vasodilation of retinal arterioles in minipigs in settings of acute BRVO. Lactate is the end metabolic product of glycolysis that, in most tissues, is produced only under anaerobic conditions and is the mediator of the hypoxia-induced vasodilation of retinal arterioles. In vivo and in vitro experiments have shown that in the retina, lactate is produced even in the presence of high oxygen concentration. 27 29  
The role of lactate in the normal retinal vascular regulation has been demonstrated in human and animal studies. Intravenous administration of lactate in healthy humans increases retinal blood flow without having a significant effect in choroidal blood flow. 2 These findings are in agreement with animal experiments in rats revealing that intravenous bolus injection and continuous infusion of lactate increases retinal blood flow. 3,4 Similarly, exercise-induced hyperlactatemia increases retinal blood flow. 30  
Changes in retinal blood flow induced by systemic administration of lactate is mainly due to increased blood velocity rather than changes in retinal vessels diameter. Indeed, Garhofer et al. 2 found no significant effect in retinal vessel diameter after intravenous infusion of lactate in healthy subjects and Brazitikos et al. 5 also showed that systemic administration or injection of lactate close to the ophthalmic circulation had no influence on retinal arterial diameters in minipigs. However, when preretinal juxta-arteriolar microinjection of l-lactate was performed, a significant and reproducible segmental dilatation of retinal arterioles in healthy retinas was observed. This vasodilation was detectable shortly after the microinjection of l-lactate, whereas microinjection of the solvent used for the preparation of the solution of lactate did not produce any detectable effect on retinal arteriolar diameter. 5 The dilation response of retinal arterioles to different concentrations of lactate was also shown recently in isolated rat retinal arterioles under normal and hypoxic conditions 7 and in isolated porcine retinal arterioles. 6 The dilation of arterioles to each concentration of lactate was developed within 20 to 30 seconds, and the highest concentration (10 mM) of l-lactate elicited nearly 70% of maximum dilatation. 6  
Indeed, it has been observed that when vasoactive drugs are injected intravenously, they do not affect the retinal vessel diameter but rather affect red blood cell velocity, as it was also the case in the study by Garhofer et al., 2 whereas when administered by the IVT route close to the study vessels, they do have an effect on the retinal vessel diameter. 5,16,17,31 These findings suggest that exposure of endothelial and/or neuronal/glial cells surrounding the retinal arteriolar wall to these agents is not sufficient to trigger a vasomotor effect, unless they reach these cells from the vitreoretinal side. 
Prostaglandins are not the principal mediators of the lactate-induced vasodilator effect, either. 5,6,32 Animal and human studies provide evidence that NO regulates retinal vascular tone in vivo 11,31 and that cells other than the endothelial cells produce and release NO in the inner retina. 11 In humans, NO contributes to the basal retinal vascular tone and is involved in flicker-induced vasodilation of the retinal vasculature. 31 In minipigs, the existence of a preretinal NO gradient from the vitreoretinal surface toward the vitreous indicates a continuous release of NO by the retinal tissue. Such a gradient is also present at a distance from visible arterioles, supporting the hypothesis that retinal cells other than vascular ones seem to release NO. 11 Furthermore, IVT juxta-arteriolar microinjection of nitro-l-arginine (l-NA), a nonspecific inhibitor of NOS, induces segmental vasoconstriction suggesting that a continuous production of NO is necessary to maintain the arteriolar tone, at least in the inner retinal vessels. 11  
Hein et al. 6 investigated the role of NO in mediating the vascular response of lactate as well as its underlying signaling mechanisms. They showed that NO is a potent mediator of lactate, as l-NAME almost completely inhibited the vasodilatory response of lactate. We have demonstrated that lactate-induced retinal arteriolar vasodilation implicates neuronal-derived NO as the major in vivo mediator in minipigs. Lactate can enter into the cells by facilitated transport via monocarboxylate transporters. 33 Such transporters are localized on endothelial and smooth muscle cells but also on the inner plasma membrane of Müller cells and on glial cells processes surrounding retinal vessels, suggesting again that neuronal cells can influence retinal vascular tone. 34 In addition, NOS isoforms are expressed in these cells, and so they have the metabolic machinery to produce NO. 35 37  
Moreover, the effect of lactate on retinal vascular tone seems to be independent of extracellular pH. Microinjections of both acid and neutral solution of l-lactate induced segmental vasodilation of similar amplitude in minipigs and microinjection of an acid solution of d-lactate caused acidification but no vasodilation. 5 These latter studies confirm that lactate induces vasodilation in retinal arterioles in in vitro and in vivo settings. In healthy retinas, IVT juxta-arteriolar injection of l-lactate 0.5 M induced a significant increase in the retinal arteriolar diameter of 27.3% ± 10%, 37% ± 5.8%, 40.9% ± 3.7%, and 40.7% ± 3.2%, respectively at 5, 10, 20, and 30 minutes after the microinjection (n = 3). 9  
In the current experimental study, we evaluated the vasomotor effect of l-lactate in acute BRVO retinas. We found a significant increase of 26.2% ± 8.2% of the arteriolar diameter 5 minutes after l-lactate juxta-arteriolar microinjection (n = 9), compared with the diameter before l-lactate microinjection. Thereafter, the vasodilatory effect of l-lactate persisted and remained significant until the end of the study period (27.7% ± 7.8% at 30 minutes, respectively). Moreover, we found no correlation between BRVO-induced arteriolar constriction at 2 hours and l-lactate-induced dilation at 30 minutes, suggesting that the extent of the initial constriction had no influence on the magnitude of the subsequent dilation. 
It should be noted that the time course of BRVO-induced constriction of the arterioles differed between the l-lactate-treated minipigs compared with the saline-treated minipigs. In the l-lactate-treated animals, arteriolar constriction did not begin until 30 minutes after the BRVO and was dilated for much of the time before that. In the saline-treated minipigs, the arterioles were only marginally dilated (∼2%) 10 minutes after BRVO. Thereafter, the vessels remained constricted. These differences can be related to the variability of the reactivity of the various retinal arterioles to laser-induced ΒRVO, to the variability of preretinal NO gradient modifications after BRVO or, most probably, to the different sample size between l-lactate-treated minipigs and saline-treated minipigs. 
It has been shown that after experimental BRVO, there is an initial increase of NO production followed by a decrease below baseline. 15 One could speculate that the less vasoconstrictory response that was observed in the l-lactate-treated minipigs 2 hours after BRVO, could imply induction of a less severe venous occlusion, with the corresponding retinal area being less affected. Retinal metabolism could have been less impaired, thus allowing a higher initial NO production (with a more potent and sustained initial arteriolar vasodilation) followed by less impairment in NO production and consequently a lesser arteriolar vasoconstriction. 
Of interest, juxta-arteriolar microinjection of l-arginine 1 mM (pH 7.4) resulted in a significant increase of 16.0% ± 3.0% and 21.0% ± 7.0% of the retinal arteriolar diameter 10 and 15 minutes, respectively, after injection, compared with the diameter before l-arginine injection, but this initial vasodilatory effect of l-arginine was transient, as it started to decrease thereafter (5.0% ± 1.5% at 30 minutes). 17 Similarly, in acute BRVO minipig retinas, 5 and 10 minutes after BQ-123 microinjection, an endothelin-1A receptor inhibitor, retinal arteriolar diameter significantly increased by 26.7% ± 7.6% and 23.7% ± 6.4%, respectively, but started to decrease thereafter (16.1% ± 3.4% at 15 minutes). 16 It appears that the vasodilator effect of l-lactate is more prolonged compared with other vasodilatory drugs that we have previously evaluated in minipig retinas with acute BRVO. 
In summary, we conducted the present study to explore the effect of l-lactate after IVT administration on the retinal arteriolar diameter using a model of experimental acute BRVO. We found that IVT juxta-arteriolar microinjection of l-lactate has a vasodilatory effect, reversing the retinal arteriolar vasoconstriction that occurs after BRVO, and that this effect persists longer when compared to l-arginine 17 and endothelin-1A receptor inhibitors. 16 Sustained-release IVT formulations of l-lactate may maintain the desired vasoactive effect and therefore significantly contribute to the restoration of normal arteriolar blood supply to the affected retina after BRVO. Maintenance of the arterial blood flow after BRVO by the use of vasodilatory drugs could prevent capillary wall collapse and could favor the development of collateral circulation, efficiently draining the area of the occluded vein and maintaining a pressure gradient sufficient for capillary perfusion, thus preventing the development of areas of capillary ischemia and its associated complications. 10 Further research is necessary toward this end. 
Footnotes
 Presented at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2010.
Footnotes
 Supported by Swiss National Science Foundation Grant 3200B0-105809.
Footnotes
 Disclosure: E. Mendrinos, None; I.K. Petropoulos, None; G. Mangioris, None; M.K. Tsilimbaris, None; D.N. Papadopoulou, None; A. Geka, None; C.J. Pournaras, None
The authors thank Nicole Gilodi and Alain Conti for technical assistance during preparation and conduct of animal experiments. 
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Figure 1.
 
Percentage change (mean ± SEM) of the retinal arteriolar diameter, measured with an RVA, after juxta-arteriolar l-lactate microinjection in experimental BRVO (n = 9). Two hours after BRVO, retinal arteriolar diameter decreased by 7.2% ± 3.3% below baseline (P = 0.004). An increase of 26.2% ± 8.2% (P = 0.004) in arteriolar diameter was evidenced 5 minutes after l-lactate juxta-arteriolar microinjection (n = 9) compared with the diameter before l-lactate microinjection. Thereafter, the vasodilatory effect of l-lactate persisted and remained significant until the end of the study period compared with the diameter before l-lactate microinjection. (26.8% ± 7.8%, P = 0.003; 27.9% ± 7.7%, P = 0.002; 27.5% ± 7.6%, P = 0.002; and 27.9% ± 7.6%, P = 0.002; and 27.7% ± 7.8%, P = 0.002 at 10, 15, 20, 25 and 30 minutes, respectively).
Figure 1.
 
Percentage change (mean ± SEM) of the retinal arteriolar diameter, measured with an RVA, after juxta-arteriolar l-lactate microinjection in experimental BRVO (n = 9). Two hours after BRVO, retinal arteriolar diameter decreased by 7.2% ± 3.3% below baseline (P = 0.004). An increase of 26.2% ± 8.2% (P = 0.004) in arteriolar diameter was evidenced 5 minutes after l-lactate juxta-arteriolar microinjection (n = 9) compared with the diameter before l-lactate microinjection. Thereafter, the vasodilatory effect of l-lactate persisted and remained significant until the end of the study period compared with the diameter before l-lactate microinjection. (26.8% ± 7.8%, P = 0.003; 27.9% ± 7.7%, P = 0.002; 27.5% ± 7.6%, P = 0.002; and 27.9% ± 7.6%, P = 0.002; and 27.7% ± 7.8%, P = 0.002 at 10, 15, 20, 25 and 30 minutes, respectively).
Figure 2.
 
Retinal arteriolar diameter changes during a typical experiment in a minipig eye. (A) Fundus photograph at baseline showing the study retinal arteriole in this experiment. (B) Site of laser photocoagulation which was performed on the retinal vein. Two hours after BRVO, there was vasoconstriction of the retinal arteriole compared with baseline, measured with the RVA. (C) Thirty minutes after a juxta-arteriolar microinjection of 50 μL l-lactate 0.5 M (pH 7.4), there was a significant vasodilation of the retinal arteriole.
Figure 2.
 
Retinal arteriolar diameter changes during a typical experiment in a minipig eye. (A) Fundus photograph at baseline showing the study retinal arteriole in this experiment. (B) Site of laser photocoagulation which was performed on the retinal vein. Two hours after BRVO, there was vasoconstriction of the retinal arteriole compared with baseline, measured with the RVA. (C) Thirty minutes after a juxta-arteriolar microinjection of 50 μL l-lactate 0.5 M (pH 7.4), there was a significant vasodilation of the retinal arteriole.
Figure 3.
 
Percentage change (mean ± SEM) of the retinal arteriolar diameter, measured with the RVA after juxta-arteriolar microinjection of the solvent (PSS) used for the preparation of l-lactate solution in experimental BRVO (n = 4). Two hours after BRVO, retinal arteriolar diameter decreased by 13% ± 3% below baseline (P = 0.026). Microinjection of 50 μL PSS (pH 7.4) did not produce any significant effect on the retinal arterioles, which remained constricted at all time-points (P > 0.1).
Figure 3.
 
Percentage change (mean ± SEM) of the retinal arteriolar diameter, measured with the RVA after juxta-arteriolar microinjection of the solvent (PSS) used for the preparation of l-lactate solution in experimental BRVO (n = 4). Two hours after BRVO, retinal arteriolar diameter decreased by 13% ± 3% below baseline (P = 0.026). Microinjection of 50 μL PSS (pH 7.4) did not produce any significant effect on the retinal arterioles, which remained constricted at all time-points (P > 0.1).
Table 1.
 
Retinal Arteriolar Diameter after IVT Juxta-arteriolar Microinjection of l-lactate in Experimental BRVO
Table 1.
 
Retinal Arteriolar Diameter after IVT Juxta-arteriolar Microinjection of l-lactate in Experimental BRVO
Time Course Retinal Diameter (AU) MAP pH Po 2 Pco 2
Baseline 210 ± 48 88.0 ± 4.4 7.38 ± 0.01 105.0 ± 1.6 39.2 ± 0.5
BRVO, 2h 195 ± 43 87.1 ± 4.5 7.38 ± 0.01 107.1 ± 1.7 39.1 ± 0.4
IVT l-lactate, 5 min 241 ± 48 86.1 ± 4.5 7.38 ± 0.02 107.4 ± 1.8 38.7 ± 0.6
IVT l-lactate, 10 min 242 ± 48 85.0 ± 4.6 7.38 ± 0.01 108.0 ± 1.7 39.7 ± 0.9
IVT l-lactate, 15 min 244 ± 45 85.6 ± 4.5 7.39 ± 0.01 107.9 ± 2.3 37.6 ± 0.3
IVT l-lactate, 20 min 243 ± 44 85.5 ± 4.5 7.39 ± 0.02 108.1 ± 2.3 38.1 ± 0.3
IVT l-lactate, 25 min 244 ± 43 85.4 ± 4.5 7.39 ± 0.02 107.5 ± 2.6 37.9 ± 0.6
IVT l-lactate, 30 min 243 ± 44 85.9 ± 4.5 7.39 ± 0.02 108.6 ± 2.2 37.8 ± 0.5
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