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1From the Departments of Physiology and 2Clinical Pharmacology, University of Vienna Medical School, Vienna, Austria; and the 3Department of Ophthalmology, University of Texas Health Science Center, San Antonio, Texas.
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Abstract |
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METHODS. In anesthetized rabbits, MAP and intraocular pressure (IOP) were measured by direct cannulation. MAP was controlled with occluders placed on the aorta and vena cava. In group 1 (n = 22), aqueous flow was measured by fluorophotometry. In group 2 (n = 21), ciliary blood flow was measured by laser Doppler flowmetry. In separate subgroups, measurements were made for 60 minutes at the control MAP of 70 mm Hg and for an additional 60 minutes at target MAPs of 80, 55, or 40 mm Hg.
RESULTS. The target MAPs achieved perfusion pressures (MAP - IOP) of 33.6 ± 1.0, 43.5 ± 0.7, 51.9 ± 0.6, and 65.2 ± 0.9 mm Hg. Ciliary blood flow was unaffected by increased perfusion pressure, but decreased progressively as perfusion pressure was lowered. Aqueous flow decreased only at the lowest perfusion pressure.
CONCLUSIONS. Under control conditions in anesthetized rabbits, aqueous production is independent of ciliary blood flow until ciliary blood flow declines below 74% of control. At ciliary blood flow below this critical level, aqueous production is blood flow dependent.
Most of the information pertinent to ciliary blood flow and aqueous production is indirect and comes from studies of ciliary ultrafiltration and pseudofacility. Those studies generally have found that moderate increases in IOP6 7 or decreases in arterial pressure8 have little effect on aqueous production—a cogent argument against ultrafiltration’s being a primary mechanism in aqueous production and against pseudofacility’s being a significant error in outflow facility measurements.9 However, the insensitivity of aqueous production to moderate decreases in perfusion pressure also suggest an insensitivity to changes in ciliary blood flow and gave rise to the textbook concept that "even though aqueous humor formation is oxygen dependent, there is an independence of aqueous flow from increments in ciliary body blood flow."2 However, there is no direct evidence to support this concept, and several reports of ciliary autoregulation indicate that perfusion pressure is an unreliable index of ciliary blood flow.10 11
The possibility that aqueous production is both dependent and independent of ciliary blood flow seems contradictory. However, in the stomach, acid secretion exhibits both types of behavior, being blood flow dependent when blood flow is reduced below a critical level and blood flow independent when blood flow is increased above that critical level.12 Recent studies showing parallel decreases in ciliary blood flow and aqueous production in response to pharmacologic vasoconstriction suggest that such a critical level also exists for the ciliary body and that ciliary blood flow below that level is inadequate to sustain aqueous production (Posey M, et al. IOVS 2003;44:ARVO E-Abstract 3435).11 13 The goal of the present study was to test the hypothesis that a relationship similar to that of gastric blood flow and acid secretion exists for ciliary blood flow and aqueous production, using mechanical manipulations of arterial pressure to vary ciliary blood flow.
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Methods |
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Animal Preparation
New Zealand albino rabbits (2–3 kg) of both sexes were housed for 1 to 3 days in the vivarium with food and water available ad libitum before the experiments. The animals were anesthetized with pentobarbital sodium (30 mg/kg, intravenously, supplemented as needed), and paralyzed with gallamine triethiodide (1 mg/kg) to eliminate eye movement. The animals were intubated through a tracheotomy and respired with room air. Expired PCO2 was monitored (Normocap 200; Datex, Tewksbury, MA) and maintained at 40 to 45 mm Hg. A heating pad was used to maintain normal body temperature (38–39°C). All intravenous injections were given through cannulae placed in the marginal ear veins.
To estimate the ocular mean arterial pressure (MAP), a catheter was inserted into the right ear artery and connected to a pressure transducer positioned at the same height above the heart as the eye. To set the MAP at the specified target pressures, hydraulic occluders were placed around the thoracic descending aorta and the inferior vena cava through a right thoracotomy. The aortic occluder was used to redirect the cardiac output to the upper body, thus increasing the MAP at the eye. The caval occluder was used to impede venous return, thus lowering cardiac output and reducing MAP throughout the circulation.
After the initial surgical preparation, the animals were mounted in a stereotaxic head holder, and the right eye was then cannulated with a 23-gauge needle inserted into the vitreous cavity through the pars plana to measure the IOP with a second pressure transducer. To avoid the rabbit ocular trauma response and release of prostaglandins, the right eye was anesthetized topically with lidocaine before the cannulation, and care was taken not to disturb the cornea and anterior chamber.14
Because of space constraints, it is not possible to measure aqueous flow and ciliary blood flow simultaneously, and so the measurements were made in separate groups of animals. It was also not possible to hold arterial pressure off baseline at several target pressures for the hour or more needed for the aqueous flow measurements, and so separate subgroups in both groups underwent an identical 1 hour of baseline measurements and 1 hour of measurements at one target pressure (i.e., 40, 55, or 80 mm Hg). A significant effort was made to ensure that the measurement conditions were as similar as possible for all animals.
Aqueous Flow Measurement
Aqueous flow was measured by fluorophotometry (FM-2; OcuMetrics, Mountain View, CA). Each animal received 4 drops of fluorescein (2.5 mg/mL, Flurox; Ocusoft, Richmond, TX) at approximately 8 AM on the day of the experiment. Two hours later, the animals were anesthetized and the treated eye was irrigated with saline to remove excess fluorescein; then the animal preparation described earlier was performed. Once the animals were mounted in the stereotaxic instrument and stable (3–3.5 hours after fluorescein application), triplicate fluorophotometric scans were performed at 15-minute intervals to measure the changes in corneal and anterior chamber fluorescein concentrations over time. Aqueous flow was calculated based on Brubaker’s method15 after applying the focal diamond correction to the raw corneal fluorescein concentration values.16 Figure 1 shows an experimental tracing and the corresponding fluorescein concentration decay curves to illustrate the aqueous flow protocol.
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To measure ciliary blood flow (Flux), the probe was attached to a modified cartridge holder of a phonograph tonearm. The tonearm counterweight was set so that the probe tip was held against the sclera with a force of approximately 0.5 g. The tonearm allowed the probe tip to move with the eye during the large changes in MAP, thereby insuring that the measurements were not influenced by changes in the force of the probe against the tissue, and that the measurements were made at the same site throughout the experiments. The probe tip was placed at a site overlying the ciliary body from which the conjunctiva had been removed. The measurement site was approximately 1 mm posterior to the limbus and was identified as the peak flow between the vessels at the limbus and the pars plana.11 13 Figure 2 shows an experimental tracing that illustrates the ciliary blood flow protocol.
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The data points in Figure 4 were fit with the following arbitrarily chosen function, where AqF is aqueous flow and CilF is ciliary flux
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Results |
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P = MAP - IOP) of 33.6 ± 1.0, 43.5 ± 0.7, 51.9 ± 0.6 and 65.2 ± 0.9 mm Hg. Table 1 shows that the baseline values in all subgroups were not significantly different and that the target MAPs for corresponding subgroups were similar and near target. The Ps were also similar for corresponding subgroups; however, the differences between the Ps at the target MAPs for subgroups 1 and 2 were statistically significant, although the differences were small (i.e., 6 and 5 mm Hg, respectively).
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Discussion |
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The present results confirm and extend an earlier report by Bill,8 who used hemorrhage to set MAP at different levels while measuring aqueous flow in anesthetized monkeys. In that study, ciliary blood flow was not measured, the IOP was held constant at 12 mm Hg, and the baseline MAPs were much higher (i.e., 119 ± 7 mm Hg) than those in the present study. Despite the differences between the studies, Bill’s finding that aqueous production in monkeys is unaffected by decreases in MAP until MAP falls below 70 to 90 mm Hg is qualitatively similar to the present results and suggests that a critical ciliary blood flow exists in other species.
Although the mechanisms responsible for this behavior are beyond the scope of the present study, the direct relation between ciliary blood flow and oxygen delivery is one plausible explanation. Oxygen is required for the optimal adenosine triphosphate (ATP) production needed to drive ciliary epithelial ionic transport. If blood flow is raised above the critical level, the excess oxygen is not extracted and simply passes through to the venous circulation. However, as blood flow is reduced below the critical level, even maximum oxygen extraction fails to provide sufficient ATP to maintain ionic transport, and so aqueous production falls. Other substrates are also possible rate-limiting candidates, but oxygen seems most likely, as it is in the stomach.12
Although the similarity of the relationships between secretion and blood flow in tissues as distinct as the ciliary body and gastric mucosa further our understanding of secretory physiology, the present results may also have clinical implications. Many of the drugs used to lower IOP in patients with glaucoma decrease aqueous production, and many of those drugs are known or potential vasoconstrictors. Other investigators have proposed that these drugs cause ciliary vasoconstriction when applied topically, which in turn decreases ultrafiltration, thereby decreasing aqueous production.18 19 Although the hydrostatic and oncotic pressure gradients across the ciliary processes argue against this proposed mechanism,1 ciliary vasoconstriction with consequent decreased oxygen delivery seems a viable hypothesis based on the present results. Moreover, in three recent studies with pharmacologic vasoconstrictors (Posey M, et al. IOVS 2003;44:ARVO E-Abstract 3435),11 13 decreases in ciliary blood flow of -21% to -37% were associated with decreases of aqueous flow of -28% to -47%. Those results also suggested a critical level of ciliary blood flow at approximately 75% of control. However, an unresolved question in those studies was whether the drugs’ effects on aqueous production were unrelated to their effect on ciliary blood flow. The present results, which were not confounded by possible direct and indirect drug effects, indicate that drug-induced ciliary blood flow reductions of those magnitudes can contribute to reductions in aqueous production.
A weakness of the present study is that it was not possible to measure ciliary blood flow and aqueous production simultaneously at all four MAPs in the same rabbit, and so division of the animals into groups with subgroups was necessary. Despite the significant care taken to achieve identical conditions across groups and to achieve the same perfusion pressures for corresponding subgroups, the two lowest perfusion pressures were approximately 5 mm Hg higher in the aqueous flow subgroups than in the ciliary blood flow subgroups. It is likely that the reported aqueous flows would have been slightly lower had their perfusion pressures been reduced an additional 5 mm Hg.
Another potential weakness of the present study is the error that could occur in fluorophotometry measurements if decreasing the perfusion pressure causes the ciliary processes to shrink, thereby delaying aqueous movement into the anterior chamber as aqueous is diverted to replace the lost volume of the processes. This potential source of error was noted by others20 as a caveat for their aqueous flow measurements after brimonidine, because they had evidence that brimonidine is a ciliary vasoconstrictor. In the case of either reduced ciliary perfusion pressure or ciliary vasoconstriction, the magnitude of this error is likely to be small, because the ciliary volume is small (
68 µL in humans15 ) and even a relatively large 20% shrinkage would be replaced within 5 minutes if aqueous production were unchanged.
An interesting finding in the present study is that ciliary blood flow was not maintained when the perfusion pressure was held for 1 hour at the two lower levels. This finding is in contrast to previous studies with this animal model, using brief (1–2 minutes) ramp increases and decreases in MAP, which elicited smaller changes in ciliary blood flow (Posey M, et al. IOVS 2003;44:ARVO E-Abstract 3435).11 13 There are several possible explanations for the different ciliary blood flow responses to brief and prolonged pressure reduction. A likely explanation is the vascular steal phenomenon whereby an autoregulatory decrease in vascular resistance in the brain shunts blood away from the eye. Another explanation is that the decline in IOP deforms the intrascleral arteries and veins, thereby increasing the resistance of the vessels supplying and draining the ciliary body. A third possibility is that the decline in IOP elicits a myogenic vasoconstriction in the ciliary body. A fourth is that the ciliary body is under baroreflex control. However, a baroreflex vasoconstriction would be likely to occur in the first few seconds after the change in pressure, which was not observed (Fig. 2) . A fifth possibility is that the systemic hypotension stimulates the production of humoral vasoconstrictors (e.g., vasopressin or angiotensin II). All these explanations are speculative, and additional studies are needed to discern which mechanisms are involved.
In summary, the present results indicate that aqueous production is blood flow independent if ciliary blood flow is above a critical level, but if blood flow is reduced below that critical level, aqueous production becomes blood flow dependent. Under control conditions in the anesthetized rabbit, that critical level occurs when ciliary blood flow is reduced below 74% of control—a blood flow reduction potentially achievable by many topical ocular antihypertensive drugs.
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Acknowledgements |
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Footnotes |
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Submitted for publication January 28, 2003; revised May 1, 2003; accepted May 11, 2003.
Disclosure: H.A. Reitsamer, None; J.W. Kiel, 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.
Corresponding author: Herbert A. Reitsamer, University of Vienna Medical School, Departments of Physiology and Clinical Pharmacology, Schwarzspanierstrasse 17, A-1090 Vienna, Austria; herbert.reitsamer{at}univie.ac.at.
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