Development of diabetes-induced acidosis in the rat retina

Abstract

We hypothesized that the retina of diabetic animals would be unusually acidic due to increased glycolytic metabolism. Acidosis in tumors and isolated retina has been shown to lead to increased VEGF. To test the hypothesis we have measured the transretinal distribution of extracellular H(+) concentration (H(+)-profiles) in retinae of control and diabetic dark-adapted intact Long-Evans rats with ion-selective electrodes. Diabetes was induced by intraperitoneal injection of streptozotocin. Intact rat retinae are normally more acidic than blood with a peak of [H(+)]o in the outer nuclear layer (ONL) that averages 30 nM higher than H(+) in the choroid. Profiles in diabetic animals were similar in shape, but diabetic retinae began to be considerably more acidic after 5 weeks of diabetes. In retinae of 1-3 month diabetics the difference between the ONL and choroid was almost twice as great as in controls. At later times, up to 6 months, some diabetics still demonstrated abnormally high levels of [H(+)]o, but others were even less acidic than controls, so that the average level of acidosis was not different. Greater variability in H(+)-profiles (both between animals and between profiles recorded in one animal) distinguished the diabetic retinae from controls. Within animals, this variability was not random, but exhibited regions of higher and lower H(+). We conclude that retinal acidosis begins to develop at an early stage of diabetes (1-3 months) in rats. However, it does not progress, and the acidity of diabetic rat retina was diminished at later stages (3-6 months). Also the diabetes-induced acidosis has a strongly expressed local character. As result, the diabetic retinas show much wider variability in [H(+)] distribution than controls. pH influences metabolic and neural processes, and these results suggest that local acidosis could play a role in the pathogenesis of diabetic retinopathy.

Keywords: Acidosis; Diabetes; Ion-selective microelectrodes; Rat; Retina; Streptozotocin; pH.

Figure 1. Converting the voltage of the H⁺-selective microelectrode (A) into a H⁺-profile (B)

A: The H⁺ voltage of the H⁺-selective microelectrode (which is the difference between the voltages of the ion-selective and reference barrels of the double barreled micropipette) consists of 840 data points obtained during 7 min of recording (2 Hz) when the electrode was continuously withdraw from the choroid to the vitreous. B: The final H⁺-profile that represents the calculated extracellular H⁺ concentration plotted against the normalized retinal depth consists of 22 data points, 20 for average [H⁺]_o in thin retinal sections (each accounting for 5% of total retinal depth), plus the average [H⁺] in the choroid and the vitreous just outside of the retina. The horizontal line marks the choroidal [H⁺]; the arrow shows the amplitude of the H⁺-profile.

Figure 2. Samples of H⁺-profiles measured in control (A–F) and diabetic (G–L) rats

Each part of the figure represents data obtained on an individual rat, and shows 3 to 6 H⁺-profiles per animal. The time after STZ injection (or vehicle injection in case of controls) is marked above each graph. Here and in all other figures [H⁺]_o is presented in nM; for reference in this figure grid lines mark corresponding pH values. The scale of the Y-axis is the same for all parts of the figure; to prevent overcrowding of the figure the labels for [H⁺]_o are shown only for parts A, D, G, and J, and the labels for pH are shown only for parts C, F, I, and L.

Figure 3. Comparison of H⁺-profile amplitudes in control (green) and diabetic (red) retinae

A: The mean values of H⁺-profile amplitudes (± s.d.) from individual rats plotted against time (in weeks) after initiation of diabetes (or injection of a vehicle for control). Green bars – controls, red bars – diabetics. Numbers of profiles averaged in each rat are marked above corresponding bars. When 2 or more data points were obtained at the same time (the same week), the corresponding bars were shifted left or right for a fraction of the week for better visual presentation. B: Comparison of the mean values (upper part) and standard deviations (lower part) of H⁺-profiles from control (green) and diabetic (red) animals. The curves (green for controls, red for diabetics) are polynomial fits of 2^nd order. Vertical dashed lines marked the borders of 3 periods: 2 – 4 weeks (less than 1 month), 5 – 13 weeks (1 – 3 months), and 14 – 30 weeks (more than 3 months). Note breaks in both Y axes (from 80 to 105 nM in the upper part of the figure and from 30 to 41 nM in the lower part of the figure) which help accommodate data points at the 5^th week.

Figure 4. Box charts of H⁺-profile amplitudes from control (green) and diabetic (red) animals

Data presented separately for 3 time periods: shorter than 1 month (on the left), 1 to 3 months (in the middle), and 3 to 6 months (on the right). ■ - the mean of profiles recorded from different rats (3 profiles per rat); ▲ – minimum;▼ – maximum; boxes cover from 10% to 90% of the values in each group; solid lines in boxes – median; dashed lines in boxes are 25% and 75%. Numbers of profiles in each group are marked near corresponding boxes.

Figure 5. Cumulative distributions of H⁺-profile amplitudes from control (green) and diabetic (red) rats

Data presented separately for 3 time periods: shorter than 1 month (on the left), 1 to 3 months (in the middle), and 3 to 6 months (on the right). On the graphs cumulative frequency is plotted against amplitude of the H⁺-profiles. The black dashed arrow in the middle graph is the KS statistic D_n1,n2; it is equal to 0.733. In order to reject the null hypothesis (control and diabetics are not different) D_n1,n2 should be larger than c(α)*√(n1+n2)/(n1*n2), where c(α) is a coefficient at a certain level of significance α (equal to 1.95 at α = 0.001), and n1 and n2 are numbers of profiles in the control and diabetic groups (15 and 30, respectively). The numerical value of c(α)*√(n1+n2)/(n1*n2) in for α=0.001 is 0.616. For these data D_n1,n2 > c(α)*√(n1+n2)/(n1*n2), i.e., the difference between diabetics and controls is statistically significant with P < 0.001.

Figure 6. Average H⁺-profiles in control (green) and diabetic (red) retinae

Control and diabetic H⁺-profiles (mean ± s.d.) for 2 time periods: 1 to 3 months (left column), and 3 to 6 months (right column). Upper row – average H⁺-profiles (mean ± s.e.m.). Numbers of individual profiles used for averaging: 1 – 3 month diabetics = 30, more than 3 month diabetics = 18, 1 – 3 month controls = 15, more than 3 month controls = 24. Lower row – normalized average H⁺-profiles, when each point is presented relative to maximum amplitude.

Figure 7. 3D presentation of [H⁺] distribution in control (A–C) and diabetic (D–F) retinae

Each part of the figure represents data obtained on individual rats, 5 to 8 H⁺-profiles per animal. The value of local [H⁺]_o was plotted against two space axes – retinal depth (in %) and eccentricity (in degrees of electrode rotation about the wall of the eye). The time after STZ injection (or vehicle injection in case of control) is marked above each graph. To emphasize the lateral gradients, the distance in the tangential direction is compressed about 4 times compared to the distance in the radial direction.