The retina is one of the most metabolically active tissues in the body. To sustain this demand in the absence of stored energy, blood supply is tightly autoregulated by local myogenic and metabolic mechanisms.1,2 Myogenic regulation refers to the adjustment of vascular resistance to maintain stable blood flow in the face of fluctuating ocular perfusion pressure. Myogenic regulation is triggered by activation of stretch-sensitive channels on the vascular endothelium3 and vascular smooth muscle cells,4,5 which stimulate the releases of vasoactive peptides to either constrict or relax smooth muscle cells.4,5 Metabolic regulation refers to the capacity for the retinal vascular bed to adjust blood supply in response to changes in local metabolic status, including oxygen and carbon dioxide tensions, pH, and the local concentrations of a range of other metabolic products.1,2,6,7
Because carbon dioxide is a major metabolic product, its local tension is known to impact local vessel tone, with hypercapnia-reducing (relative vasodilation) and hypocapnia-increasing (relative vasoconstriction) vessel tone.8–11 Changes in local carbon dioxide tension modify the balance of a range of vasoactive substances including nitric oxide,12,13 prostaglandin E2,14 and adenosine.15 Given that local carbon dioxide tension impacts vessel tone, it is likely that the capacity for the vasculature to cope with ocular perfusion pressure lowering will be affected during hypercapnia.
Studies in the cerebral circulation show that metabolic perturbation affects vasoreactivity to perfusion pressure changes. Ex vivo experiments of isolated rat diaphragmatic arterioles show that alteration of carbon dioxide levels in the perfusate significantly modifies myogenic responses to changes in intraluminal pressure.16In vivo, results show that hypercapnia shortens the range of the cerebral autoregulatory plateau in response to changes in blood pressure.17 Furthermore, in human middle cerebral arteries, hypercapnia slows their responses to a rapid change in blood pressure.18 How retinal myogenic autoregulation is modified by metabolic status has yet to be investigated.
In the present study, we hypothesized that hypercapnia would impair the vasoreactivity of retinal blood vessels to ocular perfusion pressure lowering induced by blood pressure and intraocular pressure (IOP). We tested this hypothesis in rats, where a robust myogenic autoregulatory response has been documented19 and a species increasingly used in studies of vascular physiology and ocular diseases.
Experiments were conducted in young adult Brown-Norway rats (131 ± 21 days). Rats were maintained in 22°C, 12-hour light/12-hour dark environment. Normal rat chow and water were available ad libitum. Experiments were undertaken between 8:00 AM and noon in a dedicated quiet animal surgery suite maintained at 22°C with adequate ventilation. All experimental methods and animal care procedures adhered to Association for Research in Vision and Ophthalmology's Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by Institutional Animal Care and Use Committee (Legacy Health System, Portland, OR).
Anesthesia and Surgical Preparation
General anesthesia was induced by inhalation of 2% isoflurane followed by maintenance at 1 to 2% isoflurane. The femoral arteries and veins were cannulated with polyethylene tubing (PE10 or PE50; BD Intramedic, Franklin Lakes, NJ). One femoral artery was connected to a pressure transducer (BLPR2; World Precision Instruments, Sarasota, FL) and a four-channel amplifier system (Lab-78 Trax-4/24 T; World Precision Instruments) for continuous blood pressure monitoring. A femoral vein was used for administration of drugs. The other artery or vein was used for blood drawing to induce systemic hypotension as detailed hereinafter. An orotracheal intubation was performed, and the anesthesia was switched to continuous intravenous infusion of sodium pentobarbital (2 to 5 mg kg−1 h−1, Nembutal; Oak Pharmaceuticals, Lake Forest, IL) via an infusion pump (Aladdin; World Precision Instruments). Animals were ventilated with a respirator (RSP 1002; Kent Scientific Co., Torrington, CT) with 30% oxygen at a rate between 60 and 90 breaths/min. The arterial oxygen partial pressure was maintained at an average of 110.6 ± 14.0 mmHg and a pH of 7.44 ± 0.10, as measured using a blood gas analyzer (i-STAT; Abbott Inc., Princeton, NJ). A heating pad, set at 37°C, was used to maintain body temperature throughout the experiments. Both eyes were dilated with tropicamide (0.5%; Alcon Laboratories, Inc., Fort Worth, TX) and topical anesthetized with 0.5% proparacaine hydrochloride (Bausch + Lomb, Inc., Bridgewater, NJ).
Monitoring of End-tidal Carbon Dioxide and Arterial Carbon Dioxide Partial Pressure
To avoid the need for repeated blood sampling, which makes stable continuous imaging difficult, arterial carbon dioxide partial pressure was calibrated to the end-tidal carbon dioxide level. In 12 rats, arterial carbon dioxide partial pressure ranging from 30 to 60 mmHg was measured (n = 27 samples) using the blood gas analyzer. The corresponding end-tidal carbon dioxide was recorded simultaneously using a capnometer (RSP-300; Kent Scientific Co.). The relationship between end-tidal carbon dioxide and arterial carbon dioxide partial pressure was determined using linear regression (end-tidal carbon dioxide, 0.76 mmHg; arterial carbon dioxide partial pressure, 3.5309 mmHg; R2 = 0.79). This function was used to convert end-tidal carbon dioxide to arterial carbon dioxide partial pressure.
In the current study, we set arterial carbon dioxide partial pressure in the normocapnic groups between 30 and 40 mmHg (or end-tidal carbon dioxide between 20 and 27 mmHg), which was maintained by adjusting the respiratory rate (between 60 and 90 breaths/min) and the inspiration/expiration ratio (between 15 and 45%). The arterial carbon dioxide partial pressure in the hypercapnic groups was set between 60 and 70 mmHg (or end-tidal carbon dioxide between 45 and 50 mmHg) and was achieved by adding 5% carbon dioxide to the inspired air.
Gradual Ocular Perfusion Pressure Reduction by Blood Pressure Lowering and IOP Elevation
Ocular perfusion pressure reduction was induced by blood pressure lowering and IOP elevation. For blood pressure lowering, a syringe pump was used to slowly draw blood from either a femoral artery or a vein at a rate of 0.84 ± 0.24 mL/min. Drawing started after the mean blood pressure had stabilized between 95 and 100 mmHg. The blood draw ended when blood pressure fell to between 20 and 25 mmHg. This rate of blood draw resulted in an average rate of blood pressure lowering of 10.7 ± 3.1 mmHg/min, and the average blood volume drawn was 6.0 ± 1.8 mL.
In a randomly chosen eye, gradual IOP elevation was achieved by cannulating the anterior chamber with a 33-gauge needle connected to a reservoir filled with balanced salt solution (Baxter, Deerfield, IL). To induce a gradual increase in IOP at a rate that matched the blood pressure lowering, the reservoir was raised from 10 to 70 mmHg at a rate of 9.6 ± 0.3 mmHg/min using a modified peristaltic pump (Longer Precision Pump Co., Ltd, Hebei, China). The final target IOP (70 mmHg) was confirmed using a rebound tonometer (TONOLAB, Vantaa, Finland). The ocular perfusion pressure was calculated as the difference between mean arterial blood pressure and IOP. Mean arterial blood pressure provides an estimate of ophthalmic arterial pressure, which does not require correction for hydrostatic pressure differences in rats, whereas IOP provides an estimate of retinal venous pressure.
Changes in vessel diameter in response to both methods of gradual ocular perfusion pressure reduction (blood pressure lowering and IOP elevation) were assessed in both normocapnia (control) and hypercapnia. Animals were randomly allocated to four groups (n = 7 eyes, each): normocapnia with blood pressure lowering, normocapnia with IOP elevation, hypercapnia with blood pressure lowering, and hypercapnia with IOP elevation. A minimal sample size of six was needed as determined for two groups with eight repeated measures (analysis of variance [ANOVA]) assuming an effect size of 0.5, an α value of 0.5, and a power of 0.8 (G*Power, http://www.gpower.hhu.de/).20 Two experiments were excluded, either for excessive eye movements that impacted image quality or for poor baseline blood pressure that could not be stabilized between 95 and 100 mmHg.
Vessel responses to blood pressure lowering and IOP elevation were also compared with two groups of spontaneously hypercapnic rats (n = 7 eyes for the blood pressure group and n = 5 eyes for the IOP group), in which animals were anesthetized in the same way but breathed normal room air, rather than being intubated and artificially ventilated (i.e., spontaneous hypercapnia vs. controlled hypercapnia). In these spontaneously hypercapnic animals, average arterial carbon dioxide partial pressure was 63.4 ± 11.6 mmHg, arterial oxygen partial pressure was 117.5 ± 32.58 mmHg, and pH was 7.33 ± 0.07.
Vessel Diameter Imaging and Quantification
After general preparation, rats were placed on a stage in front of a spectral-domain optical coherence tomography/confocal scanning laser ophthalmoscope (Spectralis; Heidelberg Engineering, Heidelberg, Germany). Customized rigid gas-permeable contact lenses (3.5-mm posterior radius of curvature, 5.0-mm optical zone diameter, and +5.0-diopter back vertex power) were placed on both eyes. Changes in retinal vessel diameter across a 30° field centered on the optic nerve head were recorded in infrared reflection mode (confocal scanning laser ophthalmoscope–infrared reflection) at baseline and throughout the course of ocular perfusion pressure lowering (blood pressure lowering or IOP elevation). More specifically, baseline vessel diameter was recorded over the first 20 seconds, after which ocular perfusion pressure modification was initiated and continuous recording ensued until blood pressure was decreased to 20 to 25 mmHg or IOP was increased to 70 mmHg.
Changes in vessel diameter during ocular perfusion pressure lowering were quantified using Fiji (ImageJ v2.0.0; National Institutes of Health, Bethesda, MD) as described previously.21 Image sequences were combined into a single stack and registered with custom software to allow the same regions to be analyzed across time. In each eye, three to six arterioles and three to six venules were assessed. ImageJ was used to extract the light-intensity profile perpendicularly across the vessel at one-disc diameter from the optic disc margin. Using custom-written software, vessel diameter was quantified as the distance between the two intensity troughs, which correspond to the two edges of the vessel. Changes in diameter for each vessel were expressed as a percentage relative to its own baseline. Group data were averaged into bins spanning 10 mmHg of ocular perfusion pressure.
Data Analysis and Statistics
The percentage change in vessel diameter with ocular perfusion pressure lowering (binned every 10 mmHg) in each experimental group was analyzed using one-way repeated-measures ANOVA with Dunnett's post hoc test. Differences in vessel reactivity under normocapnia and hypercapnia were compared using two-way ANOVA (ocular perfusion pressure and arterial carbon dioxide partial pressure) for blood pressure–lowering and IOP elevation challenges separately. The ocular perfusion pressure at which vessel diameter began to increase or decrease with ocular perfusion pressure lowering was determined by segmental linear regression analysis using individual diameter measurements at corresponding ocular perfusion pressure.
The critical probability (P) to reject our null hypothesis was set at 5%. Data are presented as mean ± standard deviation in the figures and within the text. All analyses were performed using Prism 7 (GraphPad Software, Inc., La Jolla, CA).
Table 1 shows baseline parameters (before ocular perfusion pressure manipulation) for the four experimental groups. The values including arterial carbon dioxide partial pressure and vessel diameters in controlled hypercapnic groups were significantly higher than the normocapnic rats (P < .05), whereas baseline ocular perfusion pressure in each group was similar.
Effects of Controlled Hypercapnia on Baseline Vessel Diameters (Before Ocular Perfusion Pressure Manipulation)
Representative baseline images in Fig. 1 show that compared with a normocapnic animal (Fig. 1A) vessels were more dilated in a hypercapnic animal (Fig. 1B). The average baseline arteriolar diameter was larger in the two hypercapnic groups (41.4 ± 5.2 μm) than in the two normocapnic groups (35.8 ± 4.5 μm). This represents a 19.6% hypercapnia-induced increase in basal arteriolar diameter (P < .001). Similarly, hypercapnia induced a 27.1% increase in venular diameter (39.5 ± 4.2 μm [normocapnia] vs. 49.8 ± 5.7 μm [hypercapnia], P < .0001). These are summarized in Table 1.
Effects of Hypercapnia on Vascular Responses to Blood Pressure Lowering
The baseline ocular perfusion pressure before blood drawing was 82.2 ± 1.4 and 83.0 ± 4.4 mmHg in the normocapnic and controlled hypercapnic groups (P = .07), respectively. Blood drawing induced similar blood pressure reductions of 70.2 ± 3.4 mmHg (from 94.1 ± 2.8 to 23.9 ± 2.3 mmHg) in the normocapnic group and 69.2 ± 5.5 mmHg (from 94.6 ± 3.0 to 22.8 ± 4.1 mmHg) in the hypercapnic group, respectively (P = .48). Under normocapnic conditions, retinal arteriolar and venular diameters remained stable with moderate ocular perfusion pressure lowering. When ocular perfusion pressure fell to 52.2 ± 1.2 mmHg for arterioles and 67.7 ± 1.7 mmHg for venules, vessel diameters started to increase steadily and reached peaks of 15.4 and 12.1% above baseline, respectively (P <.0001, one-way ANOVA for both; Fig. 2).
Under hypercapnic conditions, arteriolar and venular responses were significantly attenuated compared with normocapnia (P < .001, two-way ANOVA for both; Fig. 2). Specifically, arteriolar dilation reached a maximum of only 5.2% compared with the 15.4% seen in the normocapnic group (Fig. 2A). During hypercapnia, arteriolar diameter started to decline when ocular perfusion pressure dropped to very low levels at 23.9 ± 0.9 mmHg; this is in contrast to the dilation seen in the normocapnic group at similar ocular perfusion pressures (Fig. 2A). For venules, hypercapnia completely abolished the vasodilation seen in the normocapnic group (Fig. 2B). At a very low ocular perfusion pressure of 23.3 ± 1.5 mmHg, retinal venules showed significant constriction (P < .01, Fig. 2B).
Normocapnic and controlled hypercapnic animals were also compared against those that were naturally ventilated (spontaneously hypercapnic groups). Figs. 2A and B show that vessel responses to blood pressure lowering in spontaneously hypercapnic groups were not significantly different from those under controlled hypercapnia (P = .47 and P = .08 for arterioles and venules, respectively).
Effects of Hypercapnia on Vascular Response to IOP Elevation
The baseline ocular perfusion pressure before IOP elevation was similar in the normocapnic (86.3 ± 2.2 mmHg) and controlled hypercapnic groups (85.6 ± 3.8 mmHg, P = .13). In the normocapnic group, gradual IOP elevation from 10 to 70 mmHg induced vasodilation in both arterioles and venules. For arterioles and venules, significant dilation started when ocular perfusion pressure was lowered to 59.6 ± 2.1 and 74.6 ± 2.0 mmHg, respectively. Peak vasodilations of 11.9 and 4.5% were reached by arterioles and venules, respectively (P < .0001, Fig. 3).
Hypercapnia completely blunted both arteriolar and venular vasodilation in response to IOP elevation (P = .97 and P = .12, respectively; Fig. 3). Compared with the normocapnic groups, both arteriolar and venular responses were significantly attenuated in the controlled hypercapnia groups (P < .0001, two-way ANOVA for both; Fig. 3). At very low ocular perfusion pressures (24.3 mmHg, Fig. 3B), venules significantly constricted (P = .0002), whereas arterioles did not (P = .26). These hypercapnia data were adequately described using a single-line regression model rather than the two-line regression model needed to describe data from the normocapnic groups. Vessel response to IOP elevation in the spontaneously hypercapnic group was blunted compared with normocapnia, an effect that was similar to the controlled hypercapnic group (Fig. 3).
We evaluated the impact of altered metabolic status induced by hypercapnia on myogenic regulation in the rat retinal circulation. We found that hypercapnia significantly increased basal vessel diameter and blunted the capacity for vessels to dilate to cope with ocular perfusion pressure lowering.
Previous studies have shown that increasing arterial carbon dioxide partial pressure increases basal arteriolar diameter. The magnitude of such increases has been found to range between 1.422 and 8.5%14 in brain arterioles and from 323 to 14%24 in retinal arterioles. In the current study, arteriolar diameter was increased by 19.6% under hypercapnic conditions. A likely reason for the difference between ours and previous reports may lie in differences in induced magnitude of arterial carbon dioxide partial pressure changes.25–27 In previous studies on human subjects, arterial carbon dioxide partial pressure levels are most often mildly elevated by between 12.0 and 25.4%.23,24 In our study, direct delivery of a 5% carbon dioxide mixture by artificial ventilation raised arterial carbon dioxide partial pressure by ~70%, which would account for the larger basal dilation observed in our study.
Dissolved carbon dioxide in blood reacts with water to form carbonic acid, which is further disassociated into HCO3− and H+.28 The latter is believed to be a major mediator leading to change in vascular tone29 by stimulating release of vasoactive substances from vascular and/or glial cells.30,31 In particular, nitric oxide, which is synthesized by astrocytes,32 Müller cells,33 and endothelial cells,34 relaxes smooth muscle cells via a cyclic guanosine monophosphate signaling pathway.35 Hypercapnia may also enhance the synthesis of prostaglandin E2 through cyclooxygenase-1 and glutathione pathways,36 and it reduces calcium levels within and thus relaxes smooth muscle cells.37–39 Inhibition of prostaglandin E2 reverses hypercapnia-induced vasodilation.14
Although relaxation of smooth muscle cells is relevant to increased arteriolar diameter, it cannot explain our observation that venules dilated by 27.1% under hypercapnia, as rat venules have virtually no smooth muscle cells. Because the inner retinal circulation is a closed system, venular dilation could arise from passive distention by increased blood volume in upstream arterioles. However, we found that retinal venules dilated significantly more than arterioles (27.1 vs. 19.6%, P = .003). Differences in the way that arterioles and venules respond to stimuli have been reported in previous studies. For example, although flickering light induced dilation of retinal arteries, veins showed little change.40 In the cerebral circulation, arterioles have been shown to dilate (10%) more than venules (2%) in response to xenon gas inhalation.41 In response to hypercapnia in awake mice, cerebral arterioles dilated by 15.8%, whereas venules dilated by only 2.3%.27 These studies suggest that changes in venular diameter do not passively follow arterial changes.21
Myogenic mechanisms maintain stable blood flow over a wide range of ocular perfusion pressures, often referred to as the autoregulation plateau along the pressure flow curve.6 With extremely high or low ocular perfusion pressures (<30 mmHg), the pressure difference between tissue and intraluminal pressure results in forceful dilation and constriction, respectively. In the cerebral circulation, hypercapnia impairs vascular responses to change in perfusion pressure,42,43 as evidenced by narrowing of the autoregulation plateau.17 Dineen et al.18 showed that hypercapnia delays the initial transient hemodynamic responses to a rapid change in perfusion pressure in human middle cerebral arteries. Consistent with these studies, we showed for the first time that hypercapnia blunts the capacity of the retinal vasculature to compensate for ocular perfusion pressure lowering (Figs. 2, 3). These data support the idea that metabolic perturbation is detrimental to pressure-induced myogenic autoregulation in the retina.
Because basal vessel diameter was already increased by hypercapnia (Table 1, Fig. 1), further vasodilation in response to ocular perfusion pressure lowering might be limited by an already low myogenic tone or a reduced “vasodilatory reserve.”44 A previous study in isolated arterioles revealed that severe hypercapnia inhibits myogenic response by suppression of smooth muscle cells and activation of endothelium-dependent mechanisms.16 Hypercapnia-induced reductions in pH are also known to significantly attenuate vasodilation.45 In addition to local carbon dioxide levels, other mechanical factors are also likely to influence the retinal vasodilatory reserve.46
The results from this study highlight the importance of maintaining normal arterial carbon dioxide partial pressure levels when studying vascular physiology. As indicated by Moult et al.,47 depending on the type of anesthetic used, the respiratory system of experimental animals can be depressed to different degrees. Respiratory depression will result in accumulation of carbon dioxide. We show here that, in pentobarbital anesthetized rats spontaneously breathing room air, arterial carbon dioxide partial pressure was as high as that in rats breathing 5% carbon dioxide (63.4 vs. 64.2 mmHg). Thus, without knowing or controlling arterial carbon dioxide partial pressure, studies using spontaneous room air breath will underestimate vasoreactivity.
A number of limitations of our study are worth noting. First, in the current study, we elected to use changes in retinal vessel diameter as an index of vascular responses to ocular perfusion pressure challenge. However, the actual blood flow resulting from changes in diameter is not known. Second, although arterial carbon dioxide partial pressure was constantly adjusted to maintain levels within our target range by monitoring end-tidal carbon dioxide, arterial oxygen partial pressure was sampled only once. Although arterial oxygen partial pressure variation can influence vasoreactivity10 by ventilating animals with 30% oxygen, we believe that small variations in arterial oxygen partial pressure would not have substantially influenced our results. Finally, because IOP was continuously changing during the experiment, it was challenging to maintain a consistent focus during imaging. As a result, the image quality in experiments with IOP elevation was more variable than blood pressure–lowering manipulations.
Our current study provides evidence that alteration of metabolic status induced by hypercapnia profoundly impacts pressure-initiated myogenic autoregulation in rat retinal vessels. These findings have implication for understanding how the retinal vasculature copes with ocular perfusion pressure fluctuation in conditions such as glaucoma. Tissues that already have low oxygen or high carbon dioxide tension (which can also occur with low pH arising from increased anaerobic glycolysis) would tend to have lower vessel tone and thus reduced vasodilatory reserve. In those whose vasculature is already impaired (e.g., diabetes-induced vascular damage and arteriosclerosis with long-term systemic hypertension), a narrower autoregulatory plateau means that only a small reduction in ocular perfusion pressure would exceed the lower limit of an already compromised vasodilatory reserve.
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