Diabetes Alters pH Control in Rat Retina

Abstract

Purpose: The purpose of this study was to determine whether the ability of the rat retina to control its pH is affected by diabetes.

Methods: Double-barreled H+-selective microelectrodes were used to measure extracellular [H+] in the dark-adapted retina of intact control and diabetic Long-Evans rats 1 to 6 months after intraperitoneal injection of vehicle or streptozotocin, respectively. Two manipulations-increasing of blood glucose and intravenous injection of the carbonic anhydrase blocker dorzolamide (DZM)-were used to examine their effects on retinal pH regulation.

Results: An increase of retinal acidity was correlated with the diabetes-related increase in blood glucose, but only between 1 and 3 months of diabetes, not earlier or later. Adding intravenous glucose had no noticeable effect on the retinal acidity of control animals. In contrast, similar injections of glucose in diabetic rats significantly increased the acidity of the retina. Again, the largest increase of retinal acidity due to artificially elevated blood glucose was observed at 1 to 3 months of diabetes. Suppression of carbonic anhydrase by DZM dramatically increased the retinal acidity in both control and diabetic retinas to a similar degree. However, in controls, the strongest effect of DZM was recorded within 10 minutes after the injection, but in diabetics, the effect tended to increase with time and after 2 hours could be two to three times larger than at the beginning.

Conclusions: During development of diabetes in rats, the control over retinal pH is partly compromised so that conditions that perturb retinal pH lead to larger and/or more sustained changes than in control animals.

Figure 1

Retinal acidity plotted against blood glucose at three different periods of time after STZ or vehicle injections. Each point represents the mean amplitude of H⁺-profiles and mean blood glucose from individual control (green) and diabetic (red) rats, with standard deviations for both. Three to 10 profiles were averaged for each point. Data are grouped for three periods of time after injection of vehicle or STZ (A) shorter than 1 month (four controls and seven diabetics); (B) 1 to 3 months (five controls and 10 diabetics); and (C) 3 to 6 months (eight controls and six diabetics). The lines are linear fits for corresponding data points. The ordinate is the same for all three parts (marked on the left). The slope of the regression is significantly different from zero only for diabetics between 1 and 3 months (r² = 0.64; P = 0.0056).

Figure 2

Samples of H⁺-profiles obtained before and after artificial elevation of blood glucose in three individual rats. The panels are for a control rat 6 weeks after vehicle injection (A) and diabetics 14 weeks (B) and 20 weeks (C) after STZ injection. In panel A, three profiles were recorded before elevation of glucose (olive lines) and three were recorded at least 90 minutes after glucose elevation (light green lines). Panels B and C are similar, with red lines being profiles prior to glucose elevation and orange lines representing profiles after glucose elevation. The scales are the same in all three parts; [H⁺]_o concentration is marked in nanomoles/liter near the vertical axis in part A and the corresponding pH is marked on the right of the horizontal grid lines in part C.

Figure 3

Effect of artificial elevation of blood glucose on retinal acidity in diabetic and control rats. (A) Each point represents the amplitude of H⁺-profiles plotted against blood glucose at the time of the measurement; control rats (green) and diabetic rats (red). Filled symbols indicate before addition of glucose directly in the blood of the rat; open symbols indicate 90 or more minutes after the glucose addition. The lines (green for controls, red for diabetics) are linear fits. Squares around open circles mark data obtained from 7- to 14-week diabetics (others obtained at 3, 4, and 20 weeks). (B) The mean amplitude of H⁺-profiles plotted against mean blood glucose with corresponding standard errors for control (green) and diabetic (red) rats before blood glucose elevation (closed symbols) and after it (open symbols). Arrows point to the changed mean amplitude of H⁺-profiles and blood glucose after the manipulation.

Figure 4

Effect of artificial blood glucose elevation on retinal acidity in diabetic retinas is strongly dependent on location. (A) Three-dimensional presentation of [H⁺] distribution in one diabetic retina (14 weeks) before the glucose injection (average glucose = 335 mg/dL). The X-Y plane shows position, and the Z direction is [H⁺]. (B) [H⁺] distribution in the same retina after the glucose injection (average glucose level = 537 mg/dL). Profiles used to generate the smooth surface in (A) were recorded along a nasal-caudal line with the distance between profiles of about 170 μm (1° of eccentricity is equal to approximately 85 μm). The profiles used for part B were recorded along a line that was parallel to and about 170 μm away from the profiles in part A. The areas of spatial overlap in the lateral direction are marked by red lines on the axis.

Figure 5

Samples of H⁺-profiles obtained before and after pharmacologic suppression of carbonic anhydrase in three individual rats. The panels are for a control rat 6 weeks after vehicle injection (A) and diabetics 6 weeks (B) and 11 weeks (C) after STZ injection. Three individual profiles in each animal were recorded before application of carbonic anhydrase blocker DZM (olive lines for control and red lines for diabetics) and three profiles after DZM (light green lines for control and orange lines for diabetics). The scales are the same in all three parts; [H⁺]_o concentration is marked in nanomoles/liter near the vertical axis in panel A, and the corresponding pH is marked on the right of horizontal grid lines in panel C.

Figure 6

Effect of a carbonic anhydrase blocker on retinal acidity in diabetics and controls. (A) The descriptive statistics of retinal acidity for control (olive before DZM application; light green after DZM) and diabetic rats (red before DZM application; orange after DZM): █, the mean of H⁺-profile amplitudes; ▴, minimum; ▾, maximum; boxes cover from 25% to 75% of the values in each group; solid lines in boxes show the median. Numbers of profiles in each group are marked near corresponding boxes. (B) The floating columns represent the average acidity in the distal (left in each pair) and proximal (right in each pair) halves of the retina of control rats evoked by DZM application. The bottom of the floating columns is the average acidity before DZM application, and the top of the floating columns is the average acidity after DZM application. Three pairs of data corresponding to three individual rats; the time after injection of vehicle (in weeks) is marked under each group. (C) The same as part B for diabetics. Six pairs of data corresponding to six individual rats; the time after initiation of diabetes (in weeks) is marked under each group. Scale for the ordinate is the same as on part B (marked on the left).

Figure 7

Development of the effect of DZM on retinal acidity in time. (A) Amplitudes of H⁺-profiles in retinas of control rats after application of carbonic anhydrase blocker DZM plotted against time after DZM; three rats. The slope of the regression line was not significant (P = 0.07). (B) The same for diabetics; squares around circles mark data obtained from 6- to 8-week diabetics (others obtained on 4-, 5-, and 11-week animals); six rats. The slope of the regression line was significantly different from zero (P = 0.041).