Experimental Retinal Vein Occlusion: Effect of Acetazolamide and Carbogen (95% O₂/5% CO₂) on Preretinal PO₂

Abstract

purpose. To evaluate the variations of preretinal oxygen partial pressure (Po ₂) in normal and in ischemic postexperimental branch retinal vein occlusion (BRVO) areas, during normoxia, hyperoxia (100% O₂), and carbogen (95% O₂, 5% CO₂) breathing before and after intravenous injection of acetazolamide.

methods. Preretinal Po ₂ measurements were obtained in intervascular retinal areas, distant from the retinal vessels of 13 anesthetized mini-pigs with oxygen-sensitive microelectrodes (10 μm tip diameter) introduced through the vitreous cavity by a micromanipulator. The microelectrode tip was placed <50 μm from the vitreoretinal interface in the preretinal vitreous. Po ₂ was measured continuously for 10 minutes under systemic normoxia, hyperoxia, and carbogen breathing. A BRVO was induced with an argon green laser, and oxygen measurements were repeated under normoxia, hyperoxia, and carbogen breathing, before and after intravenous injection of acetazolamide (500 mg bolus).

results. In hyperoxia, a moderate nonsignificant preretinal Po ₂ increase in both normal (ΔPo ₂ = 2.20 ± 4.16 mm Hg; n = 25) and ischemic retinas (ΔPo ₂ = 4.30 ± 3.57 mm Hg; n = 16) was measured in spite of a substantial increase in systemic Pao ₂. Carbogen breathing induced a significant increase in systemic Paco ₂ and a higher systemic Pao ₂ than hyperoxia. Furthermore, it significantly increased the preretinal Po ₂ in normal areas (ΔPo ₂ = 19.37 ± 16.41 mm Hg; n = 26), and in ischemic areas (ΔPo ₂ = 14.94 ± 8.53 mm Hg; n = 14). Intravenous acetazolamide did not affect the preretinal Po ₂. Acetazolamide induced an increase of the preretinal Po ₂ to a greater extent when it was associated with carbogen breathing (ΔPo ₂ = 15.15 ± 9.15 mm Hg; n = 7) than when it was combined with hyperoxia (ΔPo ₂ = 6.96 ± 4.49 mm Hg; n = 7).

conclusions. Carbogen breathing significantly increased preretinal Po ₂ in normal and in ischemic postexperimental BRVO areas of mini-pigs. The concomitant use of acetazolamide injection and carbogen breathing or hyperoxia could restore an appropriate oxygenation of BRVO areas.

Branch retinal vein occlusion (BRVO) is the second most common retinal vascular disease leading to visual loss in developed countries, the most frequent cause being diabetic retinopathy. Patients in the fifth and sixth decade of life are most usually affected, and only 5% of the patients are younger than 45. ¹  

The hemodynamic modifications on the vasculature of the affected areas in acute BRVO include venous vasodilation, as well as the reduction of arteriolar blood flow. ^{2 3 4} Visual acuity is often decreased due to the development of intraretinal hemorrhages, macular edema, capillary nonperfusion, and vitreous hemorrhage secondary to retinal neovascularization. Retinal neovascularization appears in approximately 25%, ⁵ while persistent macular edema affects almost 60% of patients with BRVO. ^{6 7}  

Therefore, both the physiopathogenic mechanisms and the various treatment modalities of BRVO are important, having been in the center of clinical and experimental research. In BRVO, venous stasis induces changes in the blood–retinal barrier ^{8 9 10} and leads to extravasation and formation of extracellular retinal edema and hemorrhages. 

Arteriolar vasoconstriction, which settles in the hours after the occlusion, occurs as a result of either changes in retinal metabolism, or reduction of nitric oxide (NO) release, ¹¹ which plays a major role in retinal arteriolar tone, ¹² or myogenic vasoconstriction secondary to the intravascular pressure increase in the affected vascular bed. Reduction of arteriolar blood flow leads to tissue hypoxia in the inner retinal layers, ¹³ Na/K-ATPase pump dysfunction, formation of intracellular retinal edema, and neuronal cell destruction by necrosis and apoptosis. 

Current treatment of acute BRVO aims to restore venous circulation. Isovolemic hemodilution, ^{14 15} that leads to an increase in ocular blood flow ¹⁶ and regression of tissue hypoxia, ¹⁷ and troxerutine, ¹⁸ an erythrocyte antiaggregant, constitute currently available modalities. Their efficacy to limit visual loss has been demonstrated with varying success by randomized trials. ^{14 15 18} Grid photocoagulation improves visual prognosis in eyes with macular edema after BRVO and decreases the risk of neovascularization and vitreous hemorrhage in eyes with ischemic retinal areas larger than five disc diameters. ^{19 20}  

Pilot studies have evaluated the efficacy of fibrinolytic treatment with tissue plasminogen activator delivered by intravitreal injection, ²¹ retinal vein intravascular injection, ²² intravenous systemic injection, ²³ or superselective ophthalmic artery catheterization. ²⁴ Surgical decompression and separation of the artery and the vein by adventicectomy is an interesting approach currently under evaluation. ²⁵  

An alternative treatment aims to restore tissue normoxia by inhalation of 100% O₂ or carbogen (95% O₂, 5% CO₂). The systemic hyperoxia, thus induced, could effectively increase the oxygen partial pressure (Po ₂) of the inner retina through diffusion of oxygen from the choroid. ²⁶ Systemic hyperoxia increases the inner retinal Po ₂ to normal in retinal areas with venous stasis retinopathy as presented 48 hours after an experimental BRVO in mini-pigs. ¹³ Carbogen, through CO₂-induced ²⁷ retinal arteriolar vasodilation, might potentially be effective in increasing the diffusion of oxygen and the normalization of Po ₂ in the inner retina. Previous studies, and our preliminary results previously published, ²⁸ provided data showing that breathing carbogen induced higher preretinal Po ₂ than 100% oxygen. ^{29 30} Preretinal Po ₂ reflects oxygen diffusing from the retinal circulation. ³¹ Furthermore, the addition of intravenous administration of acetazolamide increases the Po ₂ over the optic disc in domestic pigs ³² through an increase in systemic CO₂. ³³ This effect should probably enhance the capability of hyperoxia and carbogen to induce an increase in preretinal Po ₂. 

The aim of this study was to evaluate the variations of preretinal Po ₂ in normal and in ischemic post-BRVO areas during normoxia, hyperoxia, or carbogen breathing, before and after acetazolamide administration. 

Material and Methods

Experiments were performed on one eye of 13 miniature pigs (10 to 12 kg; Arare Animal Facility, Geneva, Switzerland), whose retinas closely resemble human retina in both neuroanatomic and vascular aspects. ^{34 35} All experiments were conducted in compliance with the ARVO Statement on the Use of Animals in Research. 

Animal Preparation

Mini-pigs were prepared for experiments as previously described. ³¹ In brief, after intramuscular injection of 3 mL azaperone (Stresnil, 5 mg; Janssen Pharmaceutica, Beerse, Belgium), 2 mL of the tranquilizer midazolam maleate (Dormicum; Roche Pharma, Reinach, Switzerland; 10 mg) and 1 mL (0.5 mg) atropine, anesthesia was induced with 2–3 mg sodium thiopental (Pentothal; Abbott AG, Baar, Switzerland) injected into the ear vein. After arterial, venous, and bladder catheterization, the animal was curarized with 4 mg pancuronium bromide (Pavulon; Organon SA, Pfäffikon, Switzerland), intubated, and artificially ventilated. During the experiment, anesthesia and myorelaxation were maintained by continuous perfusion of Pentothal and Pavulon, respectively. 

Each animal was ventilated at approximately 18 strokes/min, with a continuous flow of 20% O₂ and 80% N₂O, using a variable volume respirator. Systolic and diastolic blood pressures were monitored via the femoral artery using a transducer. Paco ₂, Pao ₂, and pH were measured intermittently from the same artery with a blood gas analyzer (Labor-system, Flukiger AG, Menziken, Switzerland) and controlled by adjusting ventilatory rate, stroke volume, and composition of the inhaled gas. 

A head-holder was used to avoid respiratory movements; 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 thermo-cauterized; and an incision at the pars plana was performed. 

Po ₂ Measurements

Measurements of preretinal Po ₂ were made by double-barrel O₂-sensitive microelectrodes with a tip diameter of 10 μm, ^{36 37} as previously described. ³¹ The microelectrodes were inserted in the vitreous cavity through a sclerotomy placed 4 mm posterior to the limbus, aided by a micromanipulator ³⁸ (Fig. 1a) and positioned at a distance <50 μm from the vitreoretinal interface (Fig. 1b) . The analyzed territories were intervascular areas at a distance of at least five vessel diameters from the arterioles and far from the optic disc. In all animals, measurements were repeated in several retinal areas. 

The timeline of measurements was as follows: A baseline measurement under normoxia and a stable continuous recording for at least 10 minutes preceded inhalation of 100% of oxygen for 10 minutes. Then normoxia was induced, aiming to obtain a stable recording for at least 10 minutes; this recording was considered a baseline before inhalation of carbogen for 10 minutes. After a recovery to normoxia, a branch vein occlusion was performed by argon green laser, ^{2 3 13} inducing an ischemic microangiopathy in the studied retinal territory. In this ischemic condition, the same timeline of measurements was performed before and after intravenous injection of acetazolamide (bolus of 500 mg). 

The mean and the standard deviations (SD) of preretinal Po ₂, and systemic Pao ₂, Paco ₂, and pH were calculated at baseline and 7 minutes after starting hyperoxia or carbogen breathing. 

Statistics

A two-tailed paired Student’s t-test was used to detect differences between groups. A value of P < 0.05 was used to define statistically significant differences. For extremely small values, a conventional format of P < 0.0001 was used. A Friedman test was performed to attest the respective effect of hyperoxia and carbogen breathing in the same territory at four predetermined times (2, 5, 7, and 10 minutes). A box plot representation was used to provide an excellent visual summary of the median values and the 5%, 25%, 75%, and 95% percentiles. Moreover, a Wilcoxon signed-rank test was used to compare the effect of carbogen breathing and hyperoxia. In addition, the Bonferroni correction allowed more precise statistical analysis. 

For every presented value, the n parameter represents the number of territories where measurements were done. An n value greater than the number of mini-pigs means that more than one retinal area was analyzed in the same eye. 

Results

Under systemic normoxia (Pao ₂ = 108.43 ± 10.19 mm Hg; Paco ₂ = 35.63 ± 2.54 mm Hg; pH = 7.44 ± 0.07; n = 51), the mean preretinal Po ₂ recorded at the normal retinal intervascular areas of 13 eyes was 23.30 ± 5.26 mm Hg (n = 51), a value similar to that previously described. ^{13 31}  

Figure 2 shows a typical recording of preretinal Po ₂ in a normal retinal area in conditions of systemic normoxia followed by systemic hyperoxia, a return to baseline (i.e., normoxia) and finally carbogen inhalation. 

The inhalation of 100% O₂ induced a mean increase of preretinal Po ₂ of ΔPo ₂ = 2.20 ± 4.16 mm Hg, 13 eyes, n = 25. Under systemic hyperoxia, the mean preretinal Po ₂ increased from a mean value of 23.73 ± 5.08 mm Hg to 25.93 ± 6.26 mm Hg and that difference, although moderate, was statistically significant (P = 0.0142), yet disproportional to a substantial increase in systemic Pao ₂ (ΔPao ₂ = 299.77 ± 89.39 mm Hg). 

The inhalation of carbogen induced a mean increase of preretinal Po ₂ of ΔPo ₂ = 19.37 ± 16.41 mm Hg, 13 eyes, n = 26. The preretinal Po ₂ significantly increased from a mean value of 22.88 ± 5.50 mm Hg to 42.25 ± 16.93 mm Hg (P < 0.0001; n = 26). Under this condition, systemic Pao ₂ (ΔPao ₂ = 382.85 ± 88.12 mm Hg) and systemic Paco ₂ (ΔPaco ₂ = 13.74 ± 5.72 mm Hg) significantly increased. The CO₂ increase induced a respiratory acidosis from a mean pH value of 7.44 ± 0.07 to 7.33 ± 0.07. 

Linear regression analysis showed the variation of preretinal Po ₂ increase with time during hyperoxia and carbogen breathing (Fig. 3) . The figure reveals the statistically significant effect of carbogen breathing with time (R ² = 0.21; 13 eyes; n = 26), in contrast to hyperoxia (R ² = 0.0003; 13 eyes; n = 25). 

In addition, considering 22 retinal territories of 13 mini-pigs submitted to the same physiological conditions at four different times (2, 5, 7, and 10 minutes), the Friedman test revealed the more statistically significant effect of carbogen inhalation on the variations of preretinal Po ₂ with time (P < 0.0001; n = 22; Fig. 4 ). In contrast, during hyperoxia, the preretinal Po ₂ remained within nearly stable values, although the Friedman test revealed a moderate significant increase with time (P = 0.013; n = 22). However with the Bonferroni correction, all the tests performed for hyperoxia remained nonsignificant. 

At each of the four analyzed times, there was a significantly greater effect of carbogen inhalation on preretinal Po ₂ variations compared with systemic hyperoxia (Wilcoxon signed-rank test, P < 0.0001, n = 22). Even with the Bonferroni correction, all the tests performed for carbogen breathing remained significant, which was not the case for hyperoxia. 

In nine eyes, a branch vein occlusion was performed. Under systemic normoxia (Pao ₂ = 106.29 ± 9.11 mm Hg; Paco ₂ = 36.36 ± 2.19 mm Hg; pH = 7.46 ± 0.07; n = 25), the mean preretinal Po ₂ recorded at the affected intervascular areas was 19.41 ± 4.82 mm Hg, n = 25, a value significantly lower than that recorded before the vein occlusion in the same territories (P < 0.0001; n = 25). 

A typical recording of preretinal Po ₂ in ischemic territories under normoxia, hyperoxia, and carbogen breathing is shown in Figure 5 . 

Systemic hyperoxia induced a moderate, statistically significant elevation of preretinal Po ₂ (ΔPo ₂ = 4.30 ± 3.57 mm Hg; 9 eyes; n = 16) from a mean value of 21.51 ± 5.86 mm Hg to 25.81 ± 6.03 mm Hg (P = 0.0002; n = 16). Hyperoxia induced a similar systemic Pao ₂ change (ΔPao ₂ = 282.16 ± 94.76 mm Hg) to that reached before the BRVO. 

Carbogen breathing induced a statistically significant increase in preretinal Po ₂ (ΔPo ₂ = 14.94 ± 8.53 mm Hg; 9 eyes; n = 14) from a mean value of 20.75 ± 6.32 mm Hg to 35.69 ± 11.07 mm Hg, (P < 0.0001; n = 14). The systemic gazometric values during carbogen breathing changed in a similar way as before the BRVO (mean ΔPao ₂ = 349.36 ± 64.94 mm Hg, mean ΔPaco ₂ = 13.26 ± 6.63 mm Hg), leading to respiratory acidosis from a mean pH value of 7.43 ± 0.08 to 7.31 ± 0.08; n = 14. 

Linear regression analysis demonstrated a statistically significant increase of preretinal Po ₂ with time during carbogen breathing (R ² = 0.29; 9 eyes; n = 14), in contrast to hyperoxia (R ² = 0.024; 9 eyes; n = 16; Fig. 6 ). 

Considering 14 retinal territories of nine mini-pigs submitted to the same ischemic conditions, where Po ₂ measurements were obtained at four different times (2, 5, 7, and 10 minutes), the Friedman test revealed, as in normal retinal areas, the statistically significant effect of carbogen inhalation on the variations of preretinal Po ₂ with time (P < 0.0001; n = 14) compared to that of hyperoxia (P = 0.10; n = 14; Fig. 7 ). At each of the four analyzed times, there was a significantly greater effect of carbogen inhalation on preretinal Po ₂ variations compared with systemic hyperoxia (Wilcoxon signed-rank test, P < 0.05, n = 14). 

In ischemic retinal territories, after intravenous injection of 500 mg of acetazolamide and during normoxia, the preretinal Po ₂ values measured 7 minutes after the injection did not change significantly (ΔPo ₂ = 0.88 ± 3.14 mm Hg; 8 eyes; n = 8), from a mean value of 20.68 ± 6.73 mm Hg to 21.56 ± 6.99 mm Hg (P = 0.452; n = 8; Fig. 8a ). Sixty minutes later, the preretinal Po ₂ remained almost stable (ΔPo ₂ = 3.75 ± 4.42 mm Hg; 6 eyes; n = 6), from a mean value of 18.07 ± 5.56 mm Hg to 21.83 ± 6.33 mm Hg (P = 0.09; n = 6), although Paco ₂ increased significantly from a mean value of 36.53 ± 2.40 mm Hg to 47.74 ± 3.60 mm Hg (P = 0.0007; n = 6), simultaneously to the decrease of pH from a mean value of 7.45 ± 0.05 to 7.32 ± 0.06 (P = 0.0001; n = 6; Fig. 8b ). Pao ₂ remained within the physiological range (Pao ₂ = 107.05 ± 11.71 mm Hg; n = 6). 

The inhalation of 100% oxygen led to a moderately significant increase in preretinal Po ₂ (ΔPo ₂ = 6.96 ± 4.49 mm Hg; 7 eyes; n = 7), from a mean value of 22.71 ± 08 mm Hg to 29.67 ± 10.25 mm Hg (P = 0.006; n = 7). The Pao ₂ increase confirmed that the experiment was correctly performed (ΔPao ₂ = 329.60 ± 67.04 mm Hg). In hyperoxic conditions, pH and Paco ₂ remained practically stable (pH = 7.32 ± 0.12; Paco ₂ = 44.00 ± 4.35 mm Hg; n = 7). 

During carbogen inhalation, a significant increase in preretinal Po ₂ was recorded (ΔPo ₂ = 15.15 ± 9.15 mm Hg; 7 eyes; n = 7) from a mean value of 21.96 ± 6.36 mm Hg to 37.11 ± 12.52 mm Hg (P = 0.005; n = 7). As demonstrated in previous experiments, carbogen breathing induced a significant increase in systemic Pao ₂ (ΔPao ₂ = 376.20 ± 56.29 mm Hg), and Paco ₂ (ΔPaco ₂ = 12.01 ± 2.80 mm Hg), leading to a deeper systemic acidosis from a pH = 7.33 ± 0.05 to a pH = 7.24 ± 0.06, n = 7. 

Linear regression analysis revealed the variation of preretinal Po ₂ increase with time during hyperoxia or carbogen breathing (Fig. 9) . In those ischemic retinas and after acetazolamide injection, this test showed a statistically significant effect of carbogen with time, with R ² = 0.29 (7 eyes; n = 7), in contrast to hyperoxia with R ² = 0.098 (7 eyes; n = 7). 

Using the Friedman test, the effect of hyperoxia and carbogen breathing was analyzed at four different times (2, 5, 7, and 10 minutes) and in seven retinal territories of seven mini-pigs placed in the same ischemic conditions after acetazolamide injection (Fig. 10) . This test revealed the greater statistically significant effect of carbogen inhalation on the variations of preretinal Po ₂ with time (P = 0.0002; n = 7) than hyperoxia (P = 0.003, n = 7). Otherwise, the Wilcoxon signed-rank test demonstrated the systematically greater effect of carbogen inhalation on preretinal Po ₂ variations at all four analyzed times than in systemic hyperoxic conditions (Fig. 11) , with P < 0.05 (n = 7), except at 2 minutes (P = 0.34, n = 7). 

Discussion

In mini-pigs, an acute BRVO induces a significant decrease of preretinal Po ₂ recorded at the affected intervascular areas, a value significantly lower than that recorded before the vein occlusion in the same territories. As tissue hypoxia is established early after the occlusion as a result of the blood flow decrease, an early improvement of oxygen delivery toward the ischemic/hypoxic retinal territory has to be attempted. 

The results of our study indicated that the inhalation of carbogen could improve the delivery of oxygen to an ischemic/hypoxic retinal territory post acute BRVO, reversing tissue hypoxia. Furthermore, carbogen induced a progressive significant increase of the preretinal Po ₂ with time in normal areas (Fig. 4) . 

In normal retinas, our results confirmed previous findings in mini-pigs, indicating a regulation of the retinal blood flow during hyperoxia maintaining the preretinal Po ₂ at constant values in spite of the elevation of the systemic Pao ₂. ^{31 39} In the present series, hyperoxia induced a moderate significant increase of the preretinal Po ₂, as revealed by the Friedman test (P = 0.013; n = 22). However, after the application of the Bonferroni correction, the results obtained under hyperoxia were not statistically significant. 

Indeed, in mini-pigs and most mammals, the retinal vascularization is heterogeneous; the vascular bed of the inner retina is composed of intercommunicating capillary layers from the retinal surface to the inner nuclear layer. ^{34 35} The outer retina is not vascularized; its oxygenation is ensured by oxygen diffusion from the choroid. ^{31 40} Furthermore, the oxygen consumption of the retina is probably heterogeneous, being more important at the level of the photoreceptor inner segments, ⁴¹ as the photoreceptor expresses a higher rate of oxidative metabolism. ⁴² The heterogeneity of retinal vascularization and oxygen consumption results in intraretinal Po ₂ gradients from the surface of the retina and from the choroid toward the middle layers of the retina, which were confirmed by measurements of transretinal Po ₂ in retinas of mini-pigs ³¹ and cats. ^{41 43}  

In systemic normoxia and hyperoxia conditions, the oxygen from the choroid cannot reach the inner retina. ^{31 39 44} Hyperoxia leads to vasoconstriction of the retinal arterioles and to a decrease of the retinal blood flow of approximately 60%. ⁴⁵ In spite of the considerable increase in Pao ₂, the decrease of the arteriolar retinal blood flow and the increase of the oxygen consumption of the retina during hyperoxia ^{31 39} do not allow a supplementary contribution of oxygen delivery from the choroid to the inner retina. Thus the preretinal Po ₂ measurements reflect the oxygen diffusing from the retinal circulation. 

The preretinal Po ₂ increase, in normal areas, under carbogen breathing is due to either the effect of carbogen on the retinal circulation or the modified ability of hemoglobin (Hb) to bind oxygen as the CO₂ increase induces a rightward shift of the oxyhemoglobin dissociation curve. ⁴⁶  

Carbogen breathing induces a simultaneous increase of the systemic Pao ₂ and Paco ₂ affecting the retinal arteriolar reactivity. The elevation of Pao ₂ induces an arteriolar vasoconstriction, whereas the increase in Paco ₂ induces a vasodilation ²⁷ similar to that observed in cerebral arterioles. ⁴⁷ As a result of the vasodilatory effect on retinal arterioles secondary to the increase in Paco ₂, the inhalation of carbogen leads to a lesser reduction of retinal blood flow than that of systemic hyperoxia. ⁴⁸  

In addition to blood flow changes induced by carbogen, the CO₂ increase and the resulting pH decrease should affect the ability of Hb to bind oxygen. The blood CO₂ increase induces a rightward shift of the oxyhemoglobin dissociation curve. ⁴⁶ A rightward shift of the oxyhemoglobin dissociation curve reflects decreased affinity of Hb for oxygen, meaning that oxygen is released from Hb more readily, increasing the Pao ₂ and the oxygen availability in the tissue. 

As a result of these described effects, carbogen induced a higher increase of the Pao ₂ and preretinal Po ₂ in normal retinal areas of mini-pigs. In agreement with our findings, the contributive effect of CO_2, induces an increase in retinal juxta-arteriolar Po ₂ in rats ⁴⁴ and in normal retinas of newborn and adults rats. ³⁰ In addition, intraretinal Po ₂ profiles during carbogen breathing demonstrated that, although the oxygen supply from the choroid increases fourfold, the retinal circulation continues to provide oxygen delivery to the inner retina. ⁴⁴ These results indicate that during carbogen breathing, as in hyperoxia, ³¹ the outer retinal layers’ oxygen consumption increases and does not allow a supplementary contribution of oxygen delivery from the choroid to the inner retina. Thus the preretinal Po ₂ increase under carbogen breathing is related to oxygen diffusing from the retinal circulation. 

In ischemic postexperimental branch vein occlusion retinas, intraretinal Po ₂ gradients preserve their direction under normoxia. ¹³ Thus, the inner retina is not supplied by the O₂ diffusing from the choroid and therefore remains hypoxic. ^{13 49} The results of our study indicated that, in contrast to the inhalation of 100% of oxygen, carbogen could also improve the delivery of oxygen to an ischemic/hypoxic retinal territory post acute BRVO, leading to the restoration of appropriate oxygenation of the inner retina, reversing tissue hypoxia. 

Intravenous administration of acetazolamide increases cerebral blood flow, ⁵⁰ probably by increasing Paco ₂. ³³ This Paco ₂ increase is due to significant bicarbonate losses in the renal tubules, resulting in hyperchloremic metabolic acidosis. The CO₂ produced by cells cannot be eliminated by carbonic anhydrase and so increases Paco ₂ by diffusing through the basement membrane. ⁵¹  

In our study, in ischemic retinal territories, after intravenous injection of 500 mg of acetazolamide and during normoxia, the preretinal Po ₂ values measured continuously during 60 minutes remained almost stable, although Paco ₂ increased, concomitantly with a pH decrease (Fig. 8) . In addition, our results illustrated that concomitant administration of acetazolamide and carbogen breathing can increase preretinal Po ₂ more significantly than in association with hyperoxia. This effect is probably due to the additive effect of acetazolamide and carbogen on the elevation of Paco ₂. Indeed the systemic Paco ₂ was higher when acetazolamide was associated with carbogen than with hyperoxia (Fig. 9) . As previously mentioned, the Paco ₂ increase counterbalances the vasoconstrictive effect of hyperoxia, and decreases the ability of Hb to bind oxygen. The rightward shift of the oxyhemoglobin dissociation curve, under the effect of acetazolamide, increases the oxygen availability in the tissue. ⁵² Consequently acetazolamide has been used to modify the affinity of Hb for oxygen in the treatment of myocardial ischemia. ⁵³  

Recovery to adequate oxygenation of the retina would reflect an improvement of the retinal function. However, an increase in Paco ₂ depresses the neuronal activity of the retina leading to a shape reduction of the b wave of the ERG. ⁵⁴ This effect is due to a decrease in either saturated rod response or the b-wave amplitude ⁵⁵ as a result of the extracellular K⁺ reduction. ⁵⁶ Some recent studies indicate either an absence ⁵⁷ of effect or a reduction of contrast sensitivity under the influence of CO₂. ^{58 59}  

Taking into account those findings, the beneficial effect of an improved retinal oxygenation by carbogen inhalation with concomitant intravenous acetazolamide injection on the course of an acute BRVO remains to be demonstrated by clinical findings. 

In conclusion, our study demonstrated the efficacy of carbogen breathing to increase the preretinal oxygenation in normal retinas and to restore sufficient oxygenation of the ischemic/hypoxic retinas after BRVO. The addition of acetazolamide inducing an important elevation of Paco ₂ enhanced the effect of hyperoxia and carbogen breathing, leading to a more efficient increase in preretinal Po ₂. The Paco ₂ increase affected both the retinal circulation and the ability of Hb to bind oxygen, thus oxygen is more readily released. The beneficial effect of the concomitant administration of carbogen breathing and intravenous acetazolamide on the course of an acute BRVO remains to be demonstrated by clinical findings and functional studies. 

 Supported by Grant 32–61685.00 (CJP) from the Swiss National Research Fund (FNSR).

 Submitted for publication January 28, 2004; revised April 19, 2004; accepted April 21, 2004.

 Disclosure: J.-A.C. Pournaras, None; I.K. Petropoulos, None; J.-L. Munoz, None; C.J. Pournaras, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

(a) The micromanipulator possesses a structure facilitating the introduction of a microelectrode into the vitreous cavity through a sclerotomy and its precise three-dimensional positioning close to the vitreoretinal interface; (b) Typical positioning of a microelectrode close to the vitreoretinal interface at the time of the experimentation.

Figure 2.

 

Continuous typical recording of preretinal Po ₂ and blood gases variations under hyperoxia and carbogen breathing in a normal territory. Carbogen breathing induces a greater increase of preretinal Po ₂ (P < 0.0001; 13 eyes; n = 26) than systemic hyperoxia (P = 0.014; 13 eyes; n = 25). Both hyperoxia and carbogen induce an increase in Pao ₂, whereas carbogen breathing induces also an increase in Paco ₂ and a concomitant respiratory acidosis.

Figure 3.

 

Preretinal ΔPo ₂ variation with time in normal territories. Linear regression analysis reveals that there is no variation of preretinal ΔPo ₂ with time under hyperoxia (R ² = 0.0003), in contrast with a significant increase of preretinal ΔPo ₂ with time during carbogen breathing (R ² = 0.21).

Figure 4.

 

Boxplots displaying the ΔPo ₂ values obtained in the same 22 preretinal territories of 13 eyes at four different times (2, 5, 7, 10 minutes) under hyperoxia and carbogen breathing in normal territories. Error bars: 5% and 95% percentiles of the ΔPo ₂ values. Circles: largest or the smallest values above and below those percentiles, respectively. The Friedman test reveals the more statistically significant effect of carbogen inhalation on the variations of preretinal Po ₂ with time (P < 0.0001; n = 22) than hyperoxia (P = 0.013; n = 22). The effect of hyperoxia is not statistically significant after the application of the Bonferroni correction.

Figure 5.

 

Continuous typical recording of preretinal Po ₂ and blood gases variations under hyperoxia and carbogen breathing in a post-BRVO territory. Carbogen breathing induces a much more significant increase in preretinal Po ₂ than systemic hyperoxia. Both hyperoxia and carbogen induce an increase in Pao ₂, whereas carbogen breathing induces also an increase in Paco ₂ and a concomitant respiratory acidosis.

Figure 6.

 

Preretinal ΔPo ₂ variation with time in ischemic post-BRVO territories. Linear regression analysis reveals the variation of preretinal Po ₂ increase with time during carbogen breathing (R ² = 0.29; n = 14). During hyperoxia, the ΔPo ₂ values remain almost stable with time (R ² = 0.024; n = 16).

Figure 7.

 

Boxplots displaying the ΔPo ₂ values obtained in the same 14 preretinal territories of 9 eyes at four different times (2, 5, 7, 10 minutes) under hyperoxia and carbogen breathing in a post-BRVO territory. Error bars: 5% and 95% percentiles of the ΔPo ₂ values. Circles: largest or the smallest values above and below those percentiles, respectively. The Friedman test reveals, as in normal retinal areas, the more statistically significant effect of carbogen inhalation on the variations of preretinal Po ₂ with time (P < 0.0001; n = 14) unlike hyperoxia (P = 0.10; n = 14).

Figure 8.

 

Continuous typical recording of preretinal Po ₂ and blood gases variations under normoxia in a post-BRVO territory after i.v. acetazolamide. (a) Recording of the first 10 minutes. After a transient fall in preretinal Po ₂ due to a transient systemic hypotension, the Po ₂ values do not change significantly; (b) Recording 60 minutes later. Higher values, although not statistically significant, of preretinal Po ₂ have been reached, as Paco ₂ reaches higher values by time under the action of acetazolamide.

Figure 9.

 

Continuous typical recording of preretinal Po ₂ and blood gases variations under hyperoxia and carbogen breathing in a post-BRVO territory after i.v. acetazolamide. Carbogen breathing induces a much more significant increase in preretinal Po ₂ than systemic hyperoxia. Both hyperoxia and carbogen induce an increase in Pao ₂, whereas carbogen breathing induces also an increase in Paco ₂ leading to systemic acidosis (pH = 7.24 ± 0.06, n = 7).

Figure 10.

 

Preretinal ΔPo ₂ variation with time in ischemic post-BRVO territories after i.v. acetazolamide. Linear regression analysis shows a significant increase in preretinal ΔPo ₂ with time during hyperoxia or carbogen breathing (much more obvious during carbogen breathing), presumably under the effect of elevated Paco ₂ by acetazolamide.

Figure 11.

 

Boxplots displaying the ΔPo ₂ values obtained in the same seven preretinal territories of seven eyes at four different times (2, 5, 7, 10 minutes) under hyperoxia and carbogen breathing in ischemic post-BRVO territories after i.v. acetazolamide. Error bars: 5% and 95% percentiles of the ΔPo ₂ values. The Friedman test reveals, as in previous territories, the more statistically significant effect of carbogen inhalation on the variations of the preretinal Po ₂ with time (P = 0.0002; n = 7) than hyperoxia (P = 0.003, n = 7).

The authors thank Joël Salzmann for his editorial assistance.