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To test the hypothesis that the intravascular oxygen response to light flicker is abnormal in diabetes.
Ten eyes of normal rats and 10 eyes of rats made diabetic with streptozotocin were examined. Oxygen tension (Po2) was measured noninvasively in the retinal arteries and veins on optical section retinal images. Po2 was estimated based on the quenching by oxygen of the phosphorescence of an intravenously injected palladium porphyrin molecular probe. Measurements were conducted with and without light flicker at 10 Hz. Oxygen saturation (So2) was calculated with adjustment for the arterial pH.
In the normal rats flicker induced an increase in arterial Po2 and in the difference in arterial and venous (A-V difference) Po2 from 51 ± 5 (mean and SD) to 55 ± 7 mm Hg and from 22 ± 3 to 26 ± 5 mm Hg, respectively (P < 0.002 and 0.015, respectively). Flicker induced an increase of arterial So2 and A-V So2 difference from 64% ± 8% to 68% ± 7% and from 34% ± 4% to 38% ± 6%, respectively (P < 0.002 and 0.035, respectively). No changes in Po2 or So2 were observed with flicker in the veins. In the diabetic rats, no significant flicker-induced changes were seen in Po2 or So2 in the retinal arteries, veins, or A-V differences.
The diabetic rats lacked the flicker induced increase in arterial Po2 and So2 and also the A-V difference in Po2 and So2 observed in the normal rats. The best explanation appeared to be that diabetes impairs the increase in oxygen consumption normally provoked by light flicker.
Diabetic retinopathy is a major cause of visual loss in our society, especially in the most productive years between 20 and 65.1-4 The ophthalmoscopically visible lesions, such as microaneurysms, hemorrhages, exudates, intraretinal microvascular abnormalities, venous beading, edema, and neovascularization, indicate involvement of the retinal vasculature. In the healthy retina, retinal diameter, and blood flow are regulated by factors affecting systemic blood parameters (e.g., hypoxia,5-7 hyperoxia,5-14 hypercapnia,7,12,13,15 and hyperglycemia7,10,11,14,16) and ocular perfusion (e.g., changes in intraocular pressure,17,18 changes in systemic blood pressure5-8,14). However, diabetes has been shown to impair regulatory vessel diameter and blood flow responses.5-11,14,16,18
Some evidence indicates involvement of the retinal neural tissue, even early in the course of the disease.19-30 Light flicker offers a way to probe vascular function by manipulating neural activity. It causes an increase in retinal metabolism, especially in the inner retina.31-38 This is associated with an increase in blood vessel diameter and flow in the retina and optic nerve.32,35-37,39-51 Reduced sensitivity in flicker perimetry,23 impaired flicker-induced vascular diameter responses,52 and abnormalities in electroretinography due to light flicker20,29 have been observed in patients and animals with diabetes.
We have developed a method of measuring the oxygen tension in the retinal vessels that avoids choroidal influence.53-55 Previously, we showed that light flicker leads to an increase in the difference between the arterial and venous oxygen tension (Po2) in normal rats.38 Since no information is available on the effect of flicker on oxygen in the diabetic retinal vasculature, we tested the hypothesis that the intravascular oxygen response to light flicker is abnormal in diabetes.
Male Long Evans pigmented rats (450–650 g) were used for the study. The animals were treated in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. We studied 10 eyes of 10 normal and 10 eyes of 8 diabetic animals. Diabetes was induced by injecting 55 mg/kg of streptozotocin intravenously. Diabetic animals had blood glucose levels of 385 ± 119 mg/dL, as measured 3 to 6 days after injection; the lowest being 192 mg/dL. The studies were performed after the animals had had diabetes for 4 weeks. Both the diabetic and normal animals were studied at two or more months of age.
The rats were anesthetized with 85 mg/kg ketamine and 3.5 mg/kg xylazine administered intraperitoneally. Anesthesia was maintained by an intraperitoneal infusion of ketamine and xylazine at the rates of 0.5 mg/kg/min and 0.02 mg/kg/min, respectively. The animals breathed room air spontaneously. A femoral artery was cannulated for drawing arterial blood and monitoring the blood pressure and heart rate. These were monitored via a pressure transducer linked to a data-acquisition system (Biopac Systems Inc., Goleta, CA). Femoral arterial blood (200 μL) was drawn without air exposure, and Po2 and pH were measured with a blood gas analyzer (Radiometer, Westlake, OH). The animal holder incorporated copper tubing with flowing warm water to maintain body temperature, which was monitored via a rectal thermometer.
The pupils were dilated with 2.5% phenylephrine and 1% tropicamide. An oxygen-sensitive molecular probe, Pd-porphine (Frontier Scientific, Logan, UT), was dissolved (12 mg/mL) in physiological saline containing bovine serum albumin (60 mg/mL) buffered to a pH of 7.4, and this was injected intravenously (20 mg/kg). Hydroxypropyl methylcellulose 1% was applied to the cornea and a glass coverslip was placed on it to eliminate its refractive power and to prevent corneal dehydration. The rat was placed in front of the imaging instrument. The laser power was adjusted to 100 μW, which is safe for viewing according to the American National Standard Institute for Safety Standards.56
The principle of the imaging technique has been described in detail.53-55,57 Briefly, a diode laser beam at 532 nm was expanded, focused to a narrow line, and projected at an oblique angle on the retina after injection of the probe. A two-dimensional (2D) phosphorescence optical section retinal image perpendicular to the retinal plane was acquired by placing a near-infrared filter in front of the CCD imaging camera. The filter's transmission overlapped the phosphorescence emission of the oxygen probe. Since the incident laser beam was not coaxial with the viewing path, structures at various depths appeared laterally displaced on the phosphorescence optical section image according to their depth location. Imaging was performed at areas within 2 disc diameters (600 μm) from the edge of the optic nerve head. The intensity of the excitation laser beam and the sensitivity of the image intensifier interfaced to the imaging camera were modulated at 1600 Hz. At each location, a set of phase-delayed phosphorescence optical section images was acquired by incrementally delaying the modulated excitation laser beam with respect to the modulated imaging intensifier sensitivity.
The methodology for quantitative measurement of Po2 based on 2D phase-delayed phosphorescence intensity images has been published.54 The phase-delayed images were analyzed by using a frequency-domain approach for measurement of phosphorescence lifetime. The lifetime was used to determine the Po2 (mm Hg) according to the Stern-Volmer expression: Po2 = [(τ0/τ) − 1]/(κQ)(τ0), where τ is the phosphorescence lifetime (μs) and κQ and τ0 are the probe's quenching constant and lifetime in a 0 oxygen environment, respectively. Po2 was calculated in the major arteries and veins on the images with the use of previously published κQ and τ0 values of 381 mm Hg−1 s−1 and 637 μs, respectively.58 Arterial and venous Po2 were derived from the average of Po2 measurements in three images.
For visual stimulation, the illumination light from a slit lamp biomicroscope was used as the source for light flicker.38 A filter that transmitted light at a wavelength of 568 ± 5 nm (Edmund Industrial Optics, Barrington, NJ) was placed in the slit lamp illumination housing to eliminate overlap with the phosphorescence excitation or emission and thus to permit imaging during light flicker. The light power was 110 μW at the pupil. A shutter was attached to a solenoid and placed in front of the illumination housing of the slit lamp biomicroscope to alter the flicker frequency. The solenoid was externally driven by a square wave generator (Temma, Springboro, OH) to produce a flicker frequency of 10 Hz, which has been found to maximally stimulate vascular Po2 changes.38
The oxygen saturation (So2) of hemoglobin was calculated using the following equation59: So2 = Po2n/(Po2n + P50n), where So2 is the percentage of saturation of hemoglobin, P50 is the Po2 at which hemoglobin is half saturated, and n is the Hill coefficient.59 P50 and n were taken to be 36.0 mm Hg and 2.6, respectively, according to Cartheuser.59 The hemoglobin dissociation curve is altered by pH. Accordingly, So2 was calculated using the Bohr coefficients published by Cartheuser.59 In one rat in each group, pH data were not available. In those cases we used the mean pH value of the other rats in their groups. This seemed reasonable because the coefficients of variation of pH were 0.4% and 0.6% in the normal and diabetic rats, respectively.
The systemic data were analyzed by Student's two-tailed unpaired t-test. The Po2 and So2 data were analyzed by Student's one-tailed paired t-test, since our previous work showed that flicker will not reduce Po2.38 The results were considered significant at P ≤ 0.05.
The blood pressures in the normal and diabetic rats were 100 ± 15 (mean ± SD) and 91 ± 10 mm Hg, respectively (P = 0.07). The heart rates in the normal and diabetic animals were 222 ± 80 and 232 ± 19 beats per minute, respectively (P = 0.37). The arterial Po2 was 77 ± 11 mm Hg in the normal rats and 64 ± 12 mm Hg in the diabetic rats (P = 0.015). The arterial Pco2 was 51 ± 3.0 mm Hg in the normal rats and 53 ± 3.8 mm Hg in the diabetic rats (P = 0.22). The arterial pH in the normal and diabetic rats was 7.31 ± 0.03 and 7.31 ± 0.05, respectively (P = 0.71).
The intravascular Po2 in the normal and diabetic rats is presented in Table 1. In the normal rats, the arterial Po2 without and with flicker was 51 ± 5 and 55 ± 7 mm Hg, respectively (P = 0.002). The venous Po2 without and with flicker was 30 ± 3 and 30 ± 4 mm Hg, respectively (P = 0.42). The differences in arterial and venous (A-V difference) Po2 without and with flicker were 22 ± 3 and 26 ± 5 mm Hg, respectively (P = 0.015).
In the diabetic rats, the arterial Po2 without and with flicker was 47 ± 7 and 46 ± 7 mm Hg, respectively (P = 0.25). The venous Po2 without and with flicker was 30 ± 4 and 30 ± 3 mm Hg, respectively (P = 0.32). The A-V Po2 differences without and with flicker were 17 ± 7 and 17 ± 8 mm Hg, respectively (P = 0.40).
The intravascular So2 in the normal and diabetic rats is presented in Table 2. In the normal rats, the arterial So2 without and with flicker was 64% ± 8% and 68% ± 7%, respectively (P = 0.002). The venous So2 without and with flicker was 30% ± 6% and 30% ± 7%, respectively (P = 0.42). The A-V So2 differences without and with flicker were 34% ± 4% and 38% ± 6%, respectively (P = 0.035).
In the diabetic rats, the arterial So2 without and with flicker was 58% ± 9% and 57% ± 10%, respectively (P = 0.24). The venous So2 without and with flicker was 31% ± 8% and 31% ± 7%, respectively (P = 0.33). The A-V So2 differences without and with flicker were 27% ± 9% and 26% ± 11%, respectively (P = 0.42).
In this study, we found no detectable light flicker–induced changes in arterial or venous Po2, arterial or venous So2, or A-V difference in Po2 or So2 in diabetic rats. However, in the normal rats, light flicker induced an increase in arterial Po2 and So2 and an increase in A-V Po2 and So2 difference, as has been reported elsewhere.38 This finding confirmed our hypothesis that intravascular oxygen response to light flicker is abnormal in diabetic rats, consistent with previous reports of impaired retinal vessel autoregulation in diabetes.5-11,14,16,18 The So2 measurements in the present study were low compared with the So2 measured with spectrometry in humans.60 We attribute this largely to the hypoxic state of our rats during measurement caused by the respiratory depressant effect of the anesthetics.61
It is known that CO2 generated from respiration reduces venous pH and hence the binding of oxygen to hemoglobin. One limitation of the present study was that we had to use systemic arterial pH for all So2 calculations. Therefore, differences in pH between systemic arteries and the retinal vasculature could not be accounted for in our calculations of So2. Systemic arterial pH was essentially the same in both normal and diabetic animals, and should be very similar to retinal arterial pH near the optic disc. This factor should, therefore, not have largely affected retinal arterial So2. However, since both hyperglycemia and diabetes have been shown to reduce retinal tissue pH,62,63 venous pH in diabetic animals was probably lower than that in normal animals. Therefore, measured retinal venous Po2 would be associated with an overestimation of So2 and an underestimation of A-V So2 differences, and this phenomenon would be more prominent in the diabetic group. Nonetheless, without flicker stimulation, large A-V So2 differences existed in both groups, even though the diabetic retinas may well have been more acidic. The A-V So2 difference enlarged further during flicker in the normal group, indicating extension of the same process. Thus, it seems unlikely that pH-mediated oxygen delivery would dominate with flicker in the diabetic state if it did not dominate in the absence of flicker. It would be especially unlikely to completely eliminate enlargement of the A-V difference, as we observed. We note that any enhanced lactate production with flicker would not have had a major effect on our calculated venous So2, because lactate-induced pH changes have a much smaller, and possibly negligible, effect on hemoglobin saturation than those induced by CO2.59
One interpretation of the results is that the diabetic retina did not increase its oxygen consumption with flicker stimulation. According to the Fick principle, oxygen consumption equals the blood flow rate times the difference in concentration of oxygen between the artery and the vein.7 The ability of the diabetic retinal vasculature to regulate its blood flow in response to several stimuli has been shown to be impaired in multiple studies,5-11,14,16,18 and there is some evidence that light flicker does not increase the blood flow rate in the diabetic state as it does in normal eyes.52 Furthermore, it is known that in cats with diabetes for more than 2 years, the retina is hypoxic,64 suggestive of inadequate vascular regulation. As previously discussed, in the diabetic retina the intraretinal pH is low, which can also serve as a stimulus for compensatory regulation.63 These observations suggest that the retinal vascular compensatory mechanisms may have been stimulated to maximal capacity in the diabetic state, even before light flicker challenge. In that case, light flicker stimulation could induce no further increase in blood flow and oxygen delivery, thereby limiting oxygen consumption and enlargement of the A-V So2 difference. On the other hand, retinal venous Po2 was 30 mm Hg, indicating that at least some oxygen still may have been available for extraction, particularly since retinal cells can consume substantial oxygen even in an environment of 15 mm Hg or less.65,66 This raises an alternative possible interpretation that the diabetic state rendered the retina unable to respond to light flicker with increased oxygen consumption even though oxygen was available. This would be consistent with other evidence of abnormal neural function in diabetes.19-30
Further complementary studies are needed to clarify the roles of retinal acidity, impaired vascular regulatory responses, and intrinsic neural retinal dysfunction in response to light flicker in diabetic retinopathy. We expect that the results will aid in comprehending the pathophysiology of diabetic retinopathy and eventually lead to better treatment options.
Supported by National Institutes of Health Grants EY17918 (MS) and EY01792 (UIC), Pearle Vision Foundation, and a Research to Prevent Blindness Senior Scientific Investigator Award (MS).
Disclosure: N.P. Blair, None; J.M. Wanek, None; M. Mori, None; M. Shahidi, None