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Hyperosmolarity and electroretinogram (ERG) potentials in isolated rat retinas: possible implications in diabetic models
Experimental Eye Research 77 (2003) 115–116 www.elsevier.com/locate/yexer
Letter to the Editors
Hyperosmolarity and electroretinogram (ERG) potentials in isolated rat retinas: possible implications in diabetic models Barry S. Winkler* Eye Research Institute, Oakland University, 406 Dodge Hall, Rochester, MI 48309 4480, USA Received 3 February 2003; recieved in revised form 14 March 2003; accepted 19 March 2003
Abstract In this report, the effects of increases in the osmolarity of media superfusing isolated rat retinas on the a-wave, b-wave and oscillatory potentials of the electroretinogram (ERG) were examined. Osmolarity of the media was raised from 310 milliosmoles (control) to 340 and 370 milliosmoles by addition of NaCl or sucrose. Increases in osmolarity led to rapid decreases in the amplitudes of the b-wave and oscillatory potentials with little change in the amplitude of the a-wave. Excellent recovery of the ERG potentials was observed when control conditions were restored. The implications of these effects of an hyperosomotic load on ERG potentials in vitro are discussed with regard to a possible role of this load in models of experimental diabetes. q 2003 Elsevier Science Ltd. All rights reserved.
Li et al. (2002) have provided evidence for very early retinal damage, as monitored by ERG recordings, in a rat model of experimental diabetes. Two intravenous injections of streptozotocin separated by one week were administered to produce a severe diabetic state where the level of blood glucose was elevated, on average, to 450 mg dl21, or to 25 mM throughout the course of the experiments. They found that ‘reductions in the ERG responses (primarily the b-wave) were observed as early as 2 weeks after inducing diabetes (page 621)’. Further, they stated that ‘the damaging factor that starts to affect retinal function as early as 2 weeks after induction of diabetes does not change with time during the 25 weeks of follow-up (page 624)’ and they suggested that ‘the most probable damaging agent in the early stages of diabetes is the abnormally high level of glucose (page 623)’. They raised the possibility that the hyperglycemic condition caused an early elevation in the concentration of glutamate as a result of a decline in the activity of glutamine synthetase, the enzyme that converts glutamate to glutamine in Mu¨ller cells. In support of this, Li et al. referred to the work of Lieth et al. (1998) who reported that ‘retinal glutamate, as determined by luminometry, increased 1·6fold after 3 months of diabetes;’ this was the only time point * Dr Barry S. Winkler, Eye Research Institute, Oakland University, 406 Dodge Hall, Rochester, MI 48309 4480, USA. E-mail address: [email protected] (B.S. Winkler).
examined for this measurement in their paper. Moreover, in a subsequent paper, Lieth et al. (2000) looked for earlier diabetes-induced changes in the activity of glutamine synthetase. They found that the activity of glutamine synthetase was decreased in diabetic rat retinas after two months of diabetes, but activity was not decreased after one month. It would appear therefore that the very early onset, i.e. 2 weeks, of the diabetes-induced changes in ERG waves found by Li et al. (2002) is not caused by a decrease in the ability of retinal Mu¨ller cells to synthesize glutamine from glutamate. What then might be a cause of these extremely early diabetes-dependent alterations in ERG potentials? One consequence of the diabetic state of hyperglycemia is that the blood, ocular humors and the extracellular fluid bathing the retina are hyperosmolar. It has been known for some time that a hyperosmolar solution injected intravenously in humans (Kawasaka et al., 1977) decreases the standing potential of the eye, a potential that originates principally from the retinal pigment epithelium (Steinberg et al., 1985). Shirao and Steinberg (1987) superfused the isolated chick retina-pigment epithelium-choroid preparation with a hyperosmolar medium (25 mM mannitol added to the normal medium containing 25 mM glucose) on the retinal and/or choroidal side, and made measurements of RPE-associated potentials and resistance. Their results showed that an increase in the osmotic pressure of
0014-4835/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. DOI:10.1016/S0014-4835(03)00085-X
116
B.S. Winkler / Experimental Eye Research 77 (2003) 115–116
Fig. 1. The effect of altering the osmotic pressure of the media superfusing rat retinas on electroretinograms (ERGs) evoked by a brief, high intensity light flash. A. Osmolarity increased by 10% from 310 to 340 mOsmol. B. Osmolarity increased by 20% to 370 mOsmol. Top tracings illustrate control ERGs prior to introduction of hyperosmotic media. Middle pairs of tracings show time course of effects in response to the hyperosmolarity. Bottom pair shows recovery following return to control media. Light flash (10 msec duration) triggered the sweep of each trace. Calibration: amplitude, 100 mV; time, 20 msec.
the superfusing solution rapidly (within minutes) altered both the membrane potential and resistance of the RPE (please consult their paper for details of the rather complicated effects). However, the b-wave of the ERG was not a target of their work because as they wrote ‘a distinct b-wave does not appear (in their tracings) because of its rapid time course and relatively small amplitude (page 2019)‘. Accordingly, experiments were undertaken to examine the effects of an increase of the osmolar concentration of the oxygenated, superfusion fluid bathing isolated rat retinas on the fast (a- and b-waves) ERG potentials. Details of the composition of the media and the methods for recording these potentials in isolated rat retinas which lack the RPE can be found in Winkler, 1972, 1981. The two sides of the isolated retina were continuously superfused with the same medium. The osmotic pressure of the control medium was 310 mOsmol, and the glucose concentration was 20 mM . Two levels of hyperosmotic loads were tested: þ 10% (to 340 mOsmol) and þ 20% (to 370 mOsmol) by addition of NaCl or sucrose. Glucose concentration was kept at 20 mM in the hyperosmotic media. The effects observed were similar, irrespective of the manner in which the osmolarity was increased. Thus, it would appear that the changes in the ERG waves were produced solely by the hypertonicity of
the solution, and not by the nature of the chemical compound used to increase osmotic pressure. As shown in Fig. 1, the b-wave of the ERG was most sensitive to an increase in the osmotic pressure of the superperfusion fluid, and the effect was very fast; a substantial decrease in b-wave amplitude was observed as soon as the new solution passed by the retina. The maximum b-wave amplitude, measured from the trough of the a-wave to the peak of the b-wave, was reduced, on average ðn ¼ 5Þ by 65 and 75%, respectively, when the osmolarity of the media was raised by 10 and 20%. This effect may be due to a decrease in transretinal resistance (shrinking cells in the inner retina). During the 5-min exposure to the þ 10% hyperosmotic medium, there was generally some recovery of the amplitude of the bwave, perhaps reflecting volume regulatory mechanisms in inner retinal cells. In contrast, the amplitude of the a-wave was little changed. The oscillatory potentials were reduced in amplitude but these changes seemed smaller in comparison to the extent of reduction of the amplitude of the b-wave. When the retina was again superfused with the control solution, there was excellent recovery of the b-wave. These in vitro results clearly show that hyperosmotic loads lead to rapid changes in the amplitude of the b-wave in normal isolated rat retinas. Since the diabetic state causes a hyperosmotic load in tissues in vivo, the potential role of this load may need to be considered in assessing early changes in ERG potentials.
References Kawasaka, K., Yanagida, T., Yamamoto, S., Yonemura, D., 1977. Decrease of electrooculographic potential under osmotic stress in man. Jpn. J. Ophthalmol. 31, 23–28. Li, Q., Zemel, E., Miller, B., Perlman, I., 2002. Early retinal damage in experimental diabetes: electroretinographical and morphological observations. Exp. Eye Res. 74, 615– 625. Lieth, E., Barber, A.J., Xu, B., et al., 1998. Glial reactivity and impaired glutamate metabolism in short-term experimental diabetic retinopathy. Diabetes 47, 815–820. Lieth, E., LaNoue, K.F., Antonetti, D.A., et al., 2000. Diabetes reduces glutamate oxidation and glutamine synthetase in the retina. Exp. Eye Res. 70, 723 –730. Shirao, Y., Steinberg, R.H., 1987. Mechanisms of effects of small hyperosmotic gradients on the chick RPE. Invest. Ophthalmol. Vis. Sci. 28, 2015–2025. Steinberg, R.H., Linsenmeier, R.A., Griff, E.R., 1985. Retinal pigment epithelial cell contributions to the electroretinogram and electrooculogram. Prog. Retin. Res. 4, 33 –66. Winkler, B.S., 1972. The electroretinogram of the isolated rat retina. Vis. Res. 12, 1183–1198. Winkler, B.S., 1981. Glycolytic and oxidative metabolism in relation to retinal function. J. Gen. Physiol. 77, 667 –692.
Letter to the Editors
Hyperosmolarity and electroretinogram (ERG) potentials in isolated rat retinas: possible implications in diabetic models Barry S. Winkler* Eye Research Institute, Oakland University, 406 Dodge Hall, Rochester, MI 48309 4480, USA Received 3 February 2003; recieved in revised form 14 March 2003; accepted 19 March 2003
Abstract In this report, the effects of increases in the osmolarity of media superfusing isolated rat retinas on the a-wave, b-wave and oscillatory potentials of the electroretinogram (ERG) were examined. Osmolarity of the media was raised from 310 milliosmoles (control) to 340 and 370 milliosmoles by addition of NaCl or sucrose. Increases in osmolarity led to rapid decreases in the amplitudes of the b-wave and oscillatory potentials with little change in the amplitude of the a-wave. Excellent recovery of the ERG potentials was observed when control conditions were restored. The implications of these effects of an hyperosomotic load on ERG potentials in vitro are discussed with regard to a possible role of this load in models of experimental diabetes. q 2003 Elsevier Science Ltd. All rights reserved.
Li et al. (2002) have provided evidence for very early retinal damage, as monitored by ERG recordings, in a rat model of experimental diabetes. Two intravenous injections of streptozotocin separated by one week were administered to produce a severe diabetic state where the level of blood glucose was elevated, on average, to 450 mg dl21, or to 25 mM throughout the course of the experiments. They found that ‘reductions in the ERG responses (primarily the b-wave) were observed as early as 2 weeks after inducing diabetes (page 621)’. Further, they stated that ‘the damaging factor that starts to affect retinal function as early as 2 weeks after induction of diabetes does not change with time during the 25 weeks of follow-up (page 624)’ and they suggested that ‘the most probable damaging agent in the early stages of diabetes is the abnormally high level of glucose (page 623)’. They raised the possibility that the hyperglycemic condition caused an early elevation in the concentration of glutamate as a result of a decline in the activity of glutamine synthetase, the enzyme that converts glutamate to glutamine in Mu¨ller cells. In support of this, Li et al. referred to the work of Lieth et al. (1998) who reported that ‘retinal glutamate, as determined by luminometry, increased 1·6fold after 3 months of diabetes;’ this was the only time point * Dr Barry S. Winkler, Eye Research Institute, Oakland University, 406 Dodge Hall, Rochester, MI 48309 4480, USA. E-mail address: [email protected] (B.S. Winkler).
examined for this measurement in their paper. Moreover, in a subsequent paper, Lieth et al. (2000) looked for earlier diabetes-induced changes in the activity of glutamine synthetase. They found that the activity of glutamine synthetase was decreased in diabetic rat retinas after two months of diabetes, but activity was not decreased after one month. It would appear therefore that the very early onset, i.e. 2 weeks, of the diabetes-induced changes in ERG waves found by Li et al. (2002) is not caused by a decrease in the ability of retinal Mu¨ller cells to synthesize glutamine from glutamate. What then might be a cause of these extremely early diabetes-dependent alterations in ERG potentials? One consequence of the diabetic state of hyperglycemia is that the blood, ocular humors and the extracellular fluid bathing the retina are hyperosmolar. It has been known for some time that a hyperosmolar solution injected intravenously in humans (Kawasaka et al., 1977) decreases the standing potential of the eye, a potential that originates principally from the retinal pigment epithelium (Steinberg et al., 1985). Shirao and Steinberg (1987) superfused the isolated chick retina-pigment epithelium-choroid preparation with a hyperosmolar medium (25 mM mannitol added to the normal medium containing 25 mM glucose) on the retinal and/or choroidal side, and made measurements of RPE-associated potentials and resistance. Their results showed that an increase in the osmotic pressure of
0014-4835/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. DOI:10.1016/S0014-4835(03)00085-X
116
B.S. Winkler / Experimental Eye Research 77 (2003) 115–116
Fig. 1. The effect of altering the osmotic pressure of the media superfusing rat retinas on electroretinograms (ERGs) evoked by a brief, high intensity light flash. A. Osmolarity increased by 10% from 310 to 340 mOsmol. B. Osmolarity increased by 20% to 370 mOsmol. Top tracings illustrate control ERGs prior to introduction of hyperosmotic media. Middle pairs of tracings show time course of effects in response to the hyperosmolarity. Bottom pair shows recovery following return to control media. Light flash (10 msec duration) triggered the sweep of each trace. Calibration: amplitude, 100 mV; time, 20 msec.
the superfusing solution rapidly (within minutes) altered both the membrane potential and resistance of the RPE (please consult their paper for details of the rather complicated effects). However, the b-wave of the ERG was not a target of their work because as they wrote ‘a distinct b-wave does not appear (in their tracings) because of its rapid time course and relatively small amplitude (page 2019)‘. Accordingly, experiments were undertaken to examine the effects of an increase of the osmolar concentration of the oxygenated, superfusion fluid bathing isolated rat retinas on the fast (a- and b-waves) ERG potentials. Details of the composition of the media and the methods for recording these potentials in isolated rat retinas which lack the RPE can be found in Winkler, 1972, 1981. The two sides of the isolated retina were continuously superfused with the same medium. The osmotic pressure of the control medium was 310 mOsmol, and the glucose concentration was 20 mM . Two levels of hyperosmotic loads were tested: þ 10% (to 340 mOsmol) and þ 20% (to 370 mOsmol) by addition of NaCl or sucrose. Glucose concentration was kept at 20 mM in the hyperosmotic media. The effects observed were similar, irrespective of the manner in which the osmolarity was increased. Thus, it would appear that the changes in the ERG waves were produced solely by the hypertonicity of
the solution, and not by the nature of the chemical compound used to increase osmotic pressure. As shown in Fig. 1, the b-wave of the ERG was most sensitive to an increase in the osmotic pressure of the superperfusion fluid, and the effect was very fast; a substantial decrease in b-wave amplitude was observed as soon as the new solution passed by the retina. The maximum b-wave amplitude, measured from the trough of the a-wave to the peak of the b-wave, was reduced, on average ðn ¼ 5Þ by 65 and 75%, respectively, when the osmolarity of the media was raised by 10 and 20%. This effect may be due to a decrease in transretinal resistance (shrinking cells in the inner retina). During the 5-min exposure to the þ 10% hyperosmotic medium, there was generally some recovery of the amplitude of the bwave, perhaps reflecting volume regulatory mechanisms in inner retinal cells. In contrast, the amplitude of the a-wave was little changed. The oscillatory potentials were reduced in amplitude but these changes seemed smaller in comparison to the extent of reduction of the amplitude of the b-wave. When the retina was again superfused with the control solution, there was excellent recovery of the b-wave. These in vitro results clearly show that hyperosmotic loads lead to rapid changes in the amplitude of the b-wave in normal isolated rat retinas. Since the diabetic state causes a hyperosmotic load in tissues in vivo, the potential role of this load may need to be considered in assessing early changes in ERG potentials.
References Kawasaka, K., Yanagida, T., Yamamoto, S., Yonemura, D., 1977. Decrease of electrooculographic potential under osmotic stress in man. Jpn. J. Ophthalmol. 31, 23–28. Li, Q., Zemel, E., Miller, B., Perlman, I., 2002. Early retinal damage in experimental diabetes: electroretinographical and morphological observations. Exp. Eye Res. 74, 615– 625. Lieth, E., Barber, A.J., Xu, B., et al., 1998. Glial reactivity and impaired glutamate metabolism in short-term experimental diabetic retinopathy. Diabetes 47, 815–820. Lieth, E., LaNoue, K.F., Antonetti, D.A., et al., 2000. Diabetes reduces glutamate oxidation and glutamine synthetase in the retina. Exp. Eye Res. 70, 723 –730. Shirao, Y., Steinberg, R.H., 1987. Mechanisms of effects of small hyperosmotic gradients on the chick RPE. Invest. Ophthalmol. Vis. Sci. 28, 2015–2025. Steinberg, R.H., Linsenmeier, R.A., Griff, E.R., 1985. Retinal pigment epithelial cell contributions to the electroretinogram and electrooculogram. Prog. Retin. Res. 4, 33 –66. Winkler, B.S., 1972. The electroretinogram of the isolated rat retina. Vis. Res. 12, 1183–1198. Winkler, B.S., 1981. Glycolytic and oxidative metabolism in relation to retinal function. J. Gen. Physiol. 77, 667 –692.