The development of electroretinogram abnormalities and the possible role of polyol pathway activity in diabetic hyperglycemia and galactosemia

The Development of Electroretinogram Abnormalities and the Possible Role of Polyol Pathway Activity in Diabetic Hyperglycemia and Galactosemia Mitsuru Segawa,

Yoshihiro

Hirata, Shizuyoshi

Fujimori,

and Kodo Okada

This study examined the induction of electroretinogram abnormalities in hyperglycemia and the possible role of increased polyol pathway activity in the development of these changes. Both diabetic hyperglycemia and galactosemia caused the prolongation of peak latencies and in some cases a reduction in the amplitudes of oscillatory potentials on the b-wave. Diabetic hyperglycemia-associated abnormalities were prevented and normalized by insulin or ADN-138, an aldose reductase inhibitor. Galactosemia-induced abnormalities were inhibited by ADN-138. and were reversed either by ADN-138 treatment or by withdrawal of galactose from the diet. Polyol accumulation was prevented by insulin or ADN-138, and the elevated polyol level was reversed by insulin, ADN-138, or withdrawal of galactose in diabetic hyperglycemia and/or galactosemie. These results suggest that the increased polyol pathway activity in the hyperglycemia may be involved in the development of electroretinogram abnormalities similar to those in human diabetes: therefore, ADN-138 could be a useful drug for therapy of retinopathy in the early diabetic stage. (D1888 by Grune & Stratton. inc.

I

T HAS

BEEN generally accepted that abnormalities in the oscillatory potentials (OPs) of an electroretinogram (ERG) can be demonstrated even in the absence of ophthalmoscopically visible fundus changes in diabetes,‘,* and diabetic-like abnormalities can also be reproduced in streptozotocin (STZ)-diabetic rats.3*4 Insulin treatment has been able to correct abnormalities of OPs in both diabetic patientssV6 and rats3 On the other hand, galactosemia can reproduce diabeticlike complications in the lens,’ peripheral nerve,8 and retina.’ The role of polyol accumulation in the development of diabetic retinal microangiopathy” has been postulated; however, it has been difficult to isolate the effects of polyol accumulation from the many other metabolic alterations in diabetes. Galactosemic animals exhibiting retinal polyol accumulation” and the apparent absence of metabolic disorders commonly associated with diabetes9 may provide a valuable model for studying the influences of polyol accumulation on the biochemistry and electrophysiology of the retina. Aldose reductase inhibitors have been shown to delay cataract formation7~‘2.‘3 and to improve slowing of motor nerve conduction velocity,‘“‘6 but the influences of aldose reductase inhibitors on the neuronal components of the ERG in diabetic and galactosemic rats have not been elucidated previously. To identify the possible role of increased polyol pathway activity on the development of ERG abnormalities in diabetes, we demonstrate the induction of ERG abnormalities in both diabetic hyperglycemia and galactosemia, and the effectiveness of an aldose reductase inhibitor, ADN-138” (8’-chloro-2‘,3’-dihydrospiro[pyrroIidine-3,6’(S’H)-pyrrolo[ 1,2,3-de] [ 1,4] benzoxazinel-2,5,5’-trione) on the develop-

From the Central Research Laboratories, Kyorin Pharmaceutical Co, Ltd, Tochigi, Japan. Address reprint requests to Mitsuru Segawa. Central Research Laboratories, Kyorin Pharmaceutical Co, Ltd. 2399-01, Mitarai, Nogi-machi, Shimotsuga-gun. Tochigi, 329-01 Japan. D 1988 by Grune & Stratton, Inc. 0026-0495/88/3705-0010$03.00/0

454

ment

of ERG

abnormalities

in

both

hyperglycemia

and

galactosemia.

MATERIALS AND METHODS Animals In the experiment for diabetic hyperglycemia. male SpragueDawley rats (Charles River Japan Inc), weighing 310 g to 400 g at the beginning of the study, were fed rat chow (CE-2, Clea Japan, Inc) ad libitum. Diabetes was induced by intravenous injection of streptozotocin (Sigma, St. Louis) at a dose of 60 mg/kg. In the experiment for galactosemia, male Sprague-Dawley rats, weighing 140 g to 185 g at the beginning of the study, had a diet containing 30% galactose. In the preventive study, each medication was given for 6 weeks immediately after hyperglycemia induction, and in the therapeutic study each medication was administered from the 7th to 10th week of hyperglycemia. Zinc insulin (Novo, Tokyo; Shionogi, Osaka, Japan), 10 U per rat. was injected subcutaneously once daily, and ADN- 138 was given to diabetic rats as a 0.03% diet, and to galactosemic rats as a 0.1% diet.

ERG Recording The rats were weighed and dark-adapted for twenty minutes or more, then each rat was anesthetized with intraperitoneal injections of ketamine, 50 to 80 mg/kg (Ketalar 50, Sankyo, Tokyo), and atropine sulfate, 2 mg/kg, (Iwaki, Tokyo). Electroretinography was performed monocularly with the pupil maximally dilated by instillation of Mydrin P (Santen, Osaka, Japan). Photic stimulation by xenon lamp (3G21-P, San-ei, Tokyo) was delivered with an intensity of one joule at twenty-secondinterstimulus intervals. Using a contact lens-type electrode, ERGS evoked by strong flushes were amplified (preamplifier, AVB-10, Nihon Kohden, Tokyo) with a time constant of 0.3 second, displayed on an oscilloscope (VC-10, Nihon Kohden) and summed to five potentials using a signal averager (DAT-1100, Nihon Kohden) that also provided a copy (X,Y-recorder, WX2400, Graphtec, Tokyo) of the averaged ERG. The amplitudes of a- and b-waves were measured from the base line to the trough of the a-wave and from the trough of the a-wave to the crest of the b-wave, respectively. The amplitudes of OPs were measured by a minor modification of the method described by Algvere.’ The peak latencies were measured as the intervals between Metabolism,

Vol

37, No 5 (May). 1988: pp 454-460

ERG ABNORMALITIES AND POLYOL PATHWAY

455

Galactose-fed rats gained body weight, but compared with standard diet-fed rats, their weight was reduced by 10% to 19% at the 6th week and 16% to 20% at the 10th week. Removal of galactose from a 30% galactose diet caused a return to normal body weight by the 10th week. Cataract Formation

Fig 1. Typical wave form of averaged ERG and measurement of implicit time and amplitude. S, light stimuli; al, bl, OJ, 0.1, and OJ, peak latencies and a- and b-waves and each oscillatory potential: aa. ba. 0,a. Oza and Op. amplitudes of a- and b-waves and each oscillatory potential. Wavelet index, 0,e + 0,a + 09.

the stimulus onset and the peak of the corresponding a- and b-waves and OPs. OPs were designated as 0,, Or, and 0, in order of superimposition on the b-wave, and the sum of these amplitudes was expressed as the wavelet index.’ (Fig 1). Sugar Estimation Retinas were carefully removed, weighed, and boiled for twenty minutes in 1.0 mL of distilled water containing a-methyl-Dmannoside (Tokyo Kasei, Tokyo) as the internal standard. The supernatants deproteinized by the method of Somogyi” were lyophilized. myo_Inositol and sugars in the freeze-dried retina extracts were converted into their trimethylsilyl ethersI Samples were run on a gas chromatograph, GC-7AG, (Shimadzu, Kyoto, Japan) equipped with a flame-ionization detector. The chromatographic column was a glass column, 1.1 m x 2.6 mm inner diameter, packed with 2% OV-101 on loo-120 mesh Chromosorb W (HP). The detector temperature was 250°C and the flow rate of Helium carrier gas was 50 mL/min. Blood glucosezOand galactose*’ were measured enzymatically. Sfatistical Analysis Data values are presented as mean f SEM and significance levels were estimated using the Wilcoxon rank sum test for unpaired data (two-sided). Linear regression was calculated by the least-squares method. A P value of d.05 was regarded as being statistically significant.

The eyes were examined without magnification when electroretinography was performed. Cataracts developed in approximately 30% of diabetic rats’ eyes at the 6th week, and in 100% at the 10th week. Insulin or ADN- 138 treatment completely prevented cataract formation at the 6th week (the preventive study). At the 10th week, almost all eyes of ADN-138-treated rats developed lenticular opacities, but insulin therapy stopped any further increase in the rate of cataract formation (the therapeutic study). On the other hand, cataracts were detected at the 6th and 10th weeks in all galactose-fed rats. No lenticular opacities were observed at the 6th week in rats receiving ADN-138 (the preventive study), but neither ADN-138 treatment nor galactose withdrawal ameliorated the cataracts already developed at the 10th week (the therapeutic study). Eiectroretinogram

Data for both diabetic and galactosemic animals are summarized in Tables 1 and 2. There was no significant difference in amplitudes and latencies of the a- and b-waves in either group. Typical ERG abnormalities induced by hyperglycemia were mainly the prolongation of peak latenties and the reduction of amplitudes of OPs on the ascending branch of the b-wave (Tables 1 and 2). Because of the infrequency of amplitude reduction, our study focused on the peak latencies of OPs. Diabetic hyperglycemia produced the prolongation of peak latencies of OPs at the 6th and 10th weeks compared with normoglycemia, but there was no further prolongation between the 6th and 10th weeks. The prolongation of peak latencies of OPs was prevented by treatment with insulin or

RESULTS

Blood Glucose, Galactose Concentrations, and Body Weight F : Fructose

In rats with STZ-induced diabetes, at the 6th week blood glucose concentrations, in mg/lOO mL, were 454 f 18 for

diabetic rats, 78 + 13 for insulin-treated rats, and 421 * 16 for ADN-138treated rats; at the 10th week, these values were 466 * 7 for diabetic rats, 61 2 16 for insulin-treated rats, and 37 1 f 13 for ADN- 138-treated rats. Diabetic and ADN- 138-treated rats had 12% to 22% body weight loss at the 6th week and 6% to 12% at the 10th week; no body weight loss occurred in insulin-treated rats in either the preventive or the therapeutic study. In rats fed galactose, blood glucose and galactose concentrations were, in mg/ 100 mL, 128 +- 5 and 39 * 12 at the 6th week and 109 k 9 and 67 * 13 at the 10th week; no changes in blood glucose or galactose concentrations were observed between galactose-fed rats and ADN-138-treated rats.

S

: Sorbitol

M :

myo-Inositol

\

1

CONTROL NORMOGLYCEMIA DIRBETIC

iKi::::

ADN-138 b.OI%CILI

HYPERGLYCFMIA

Fig 2. Inhibitory effects of insulin and ADN-138 on polyol accumulation in diabetic rats (N = 4 to 10). Each bar represents mean + SEM: l P < .05 Y normoglycemic control; # P c .05 v hyperglycemic control.

study

6.93

7

10

e 0.176

f 0.188

z 0.168

f 0.112

* 0.108

t 0.256

+ 0.055

? 0.735

al

6.49 6.62

6

7

10

10

10

I”S”hl

ADN-138

e 0.166

t 0.150

6.79tO.117

6.44

7

7

6.44

7

6

6.65

6.40

7.08

COtW0l

Hwerglycsmla

NCWlogl”WlWl

10

4

9

0

6

ADN-138

study

6

Therape”tlc

6

6.46

9

ln*ulin

6.31

9

6

0‘ Rats

0

COllWOl

Hyperglycemia

Namoglycemla

Preventive

Number

58.5

55.4

58.9

60.7

51.8

57.9

60.9

52.8

55.0

57.0

54.0

1.49

1.42

+ 2.09

‘- 3.30

+ 3.09

+ 1.60

+ 1.8,

+ 2.09

+ 1.3,

+ 3.66

*

f

+ 2.05

bl

18.6

18.0

20.2

17.2

17.6

17.9

18.1

17.8

19.9

17.7

18.0

011

Time

i 0.149-t

t 0.522t

t 0.36,.

e 0.148

t 0.176

f 0.169

+ 0.169t

+ 0.472t

+ 0.440.

r 0.137

+ 0.123

Imphat

imY)cl

26.7

25.4

28.5

25.1

25.6

26.3

25.6

25.3

28.6

25.2

26.1

* 0.162-t

f 0.5937

+ 0.603.

+ 0.358

t 0.186

t 0.256

f 0.280t

* 0.421t

+ 0.723.

e 0.208

e 0.141

021

38.1

38.1

37.1

41.0

37.0

38.6

37.0

36.9

36.9

4.09

38.0

1.08’

1.68

1.12.

+ 0.482-t

r 0.503t

t

+ 0.362

t 0.622

t

e 0.609t

f 0.11st

*

2 0.434

+ 0.251

011

,040

,132

746

,224

,216

,211

1293

1392

1181

1272

+ 0,a

Index

73.3

r 95.9

+ 65.0

+ 99.9’

+ 91.7

* 69.8

f

e 60.2

+ 52.5

* 80.9

* 68.6

+ 74.9

+ 0,a

1380

0,a

w&t

733

509

544

460

613

653

680

80,

731

498

655

l&Iv)

19.5

57.3

f

21.8

+ 26.7

t

+ 28.2

+ 25.3

e 17.4

+ 22.7

*

e 37.2

+ 20.0

r 30.1

aa

Ampliiude

Table 1. Preventive and Therapeutic Effects of Insulin and ADN-138 on the Prolongation of Peak Latencies of Oscillatory Potentials in Diabetic Rats

,366

,035

1072

898

,161

,212

1205

,283

,472

,120

1360

56.2

74.1

f 64.4

e 56.8

+ 81.8

+ 49.7

+ 36.1

t

+ 50.7

+ 51.7

+ 63.2

t

+ 75.3

ba

ERG ABNORMALITIES

AND POLYOL PATHWAY

457

D M

: :

Dulcitol myo-Inositol

I

CONTROL NORMOGLYCEMIA

ADN-138 I.l%DICI

,,ilill~I1AhAI

/ ALATIOYMIA

Fig 3. Effects of insulin and ADN-138 in decreasing elevated polyol contents in diabetic rats (N = 6 to 71. Each bar represents mean k SEM: l P < .05 Y normoglycamic control: # P -c .05 Y hyperglycemic control.

ADN-138 for 6 weeks (Table l), and the already prolonged latencies at the 6th week were normalized by insulin and reduced by ADN-138 treatment for 4 weeks (Table 1). On the other hand, galactosemia also prolonged peak latencies of OPs at the 6th and 10th weeks to levels similar to those in diabetic hyperglycemia (Table 2), and there were no further increases in peak latencies between the 6th and 10th weeks. The prolongation of peak latencies was inhibited by treatment with ADN-138 for 6 weeks (Table 2) and the already established deficit at the 6th week was normalized by ADN-138 and reduced by galactose removal from the diet at the 10th week (Table 2). Retinal Polyol Levels

Changes in retinal polyol levels are presented in Figs 2 and 3 (diabetic hyperglycemia) and in Figs 4 and 5 (galactosemia). In S’TZ-diabetic rats, six weeks after hyperglycemia induction, the retinal glucose, sorbitol, and fructose contents were increased but myo-inositol was not affected as compared with normoglycemia (Fig 2). Insulin prevented the elevation of retinal glucose, sorbitol, and fructose levels depending on the blood glucose levels, and ADN-138 reduced the sorbitol and fructose accumulations by 95% and

Effects of ADN-138 and withdrawal of galactosa in decreasing elevated sugar contents in galactosemic rats (N = 7 to 6). Each bar represents mean + SENT, l P < .05 Y normoglycamic control; # P -c .05 v galactosamic control.

76%, respectively, without affecting glucose levels in the retina and blood at the 6th week (Fig 2). Ten weeks later, diabetic hyperglycemia maintained the elevated glucose, fructose, and sorbitol levels but did not affect myo-inositol in the retina by comparison with normoglycemia. Insulin normalized the elevated glucose, fructose, and sorbitol levels in the retina, and ADN- 138 resulted in normal sorbitol content and a 59% reduction in accumulated fructose despite elevated glucose levels in the retina and blood at the 10th week (Fig 3). Moreover, there was a significant correlation between the prolongation of peak latencies of OPs and the accumulation of sorbitol and fructose in the retina (r = .770 and ,782, respectively; P < .Ol). In galactose-fed rats, 6 weeks after galactosemia induction, the retinal galactose and dulcitol contents were increased without affecting myo-inositol content compared to normoglycemia (Fig 4). ADN-138 inhibited the dulcitol accumulation by 85% in the retina but did not affect galactose levels in the retina and serum at the 6th week (Fig 4). Ten weeks later, galactosemia maintained the increased galactose and dulcitol contents but did not affect myoinositol in the retina (Fig 5). ADN-138 produced an 81% reduction in the accumulated dulcitol content in the retina without affecting galactose levels in the retina and serum (Fig 5), and galactose removal from the diet normalized galactose and dulcitol levels in the retina as indicated by galactose elimination from blood at the 10th week (Fig 5). These results revealed a significant correlation between the prolongation of peak latencies of OPs and dulcitol accumulation in the retina (I = .862, P -c .Ol). DISCUSSION

Fig 4. Inhibitory affect of ADN-138 on dulcitol accumulation in galactosamic rats (N = 7). Each bar represents mean + SEM; l P < .05 v normoglycamic control; # P < .05 v galactosamic control.

This study demonstrates that both diabetic hyperglycemia and galactosemia result in the development of ERG abnormalities, and that these changes are not only prevented but also restored by normalization of retinal polyol accumulation in the early hyperglycemic stage. Galactosemia can develop lenticular opacity,7 slowing of motor nerve conduction velocity,* and retinal vascular lesions9 similar to those in diabetic hyperglycemia. ERG abnormalities in OPS’*~and the c-wave** have been reported previously in STZ-diabetic rats, but not in galactosemic rats. In this study, galactosemia also induced the

8r.3

slprssse*

as mean

tP

< .05

vgalactosemic

lP < .05 vnamoglycemie.

Values

Namcdycsm~a

Study

6.33 6.1,

7

7

8

7

8

10

10

10

10 6.40

6.28

6.26 6.39

7

0

f 8EM.

control.

6.54 6.36

6

7

Therapeutic

7

ADN-138

6.29 6.2,

7

7

0‘ fiats

ClJdd

Galactnsamia

W&

Number

i 0.140

+ 0.0685

k 0.135

t 0.129

t 0.0746

r 0.0574

+ 0.144

+ 0.161

+ 0.1079

t 0.154

al

58.4

55.6

58.6

56.2

1.93

+ 1.78

t

+ 2.44

e 2.74

i 2.17 * 2.1,

52.7

* 2.07

+ 0.8,

t 2.3,

+ 1.62

67.0

53.3

61.8

54.5

66.5

bl

17.9

17.3

18.8

17.3

17.6

19.5

16.8

19.4

0.2927

+ 0.177t

f

+ 0.,7,*

+ 0.16,

+ 0.244

+ 0.139

f 0.215,

* 0.350.

+ 0.147 t 0.133

18.2

Tmw

17.0

011

lmplicot

bwecl

26.3

25.3

27.3

24.9

25.3

26.0

24.3

27.9

24.5

25.8

2 0.296.t

+ 0.510t

e 0.237.

+ 0.317

+ 0.400

T 0.292

+ 0.309,

+ 0.468.

+ 0.182

+ 0.127

D,l

37.7

40.1

38.8

42.7

36.9

37.0

36.5

36.3

44.4

36.3

1.14

0.776.

f 0.569-t

* 0.492-t

t

e 0.429

+ 0.628

+

+ 0.584t

* 0.937.

+ 0.441

+ 0.476

01,

,157

,350

962

,259

,410

1380

1630

,017

1477

86.1

+ 0,a

Index

93.0

155.

91.2

+ 72.8

+ 9,.9t

f

f

* 91.5

f

+ 75.7t

+ 88.7.

e 116

f

+ 0,a

,260

0,a

wavelet

61,

679

790+

665

670

919

749

473

719

748

CV)

18.3

19.2

113

39.4

+ 45.0

+ 36.9

f

f 42.0

t

f

2 48.3

+ 24.3

e 69.3

M

Amplitude

,069

,194

998

1290

,320

14.2

f

59.4 + 66.0

f

+ 105

+ 71.5

+ 63.8

51.5

e 60.6

+ 47.9

t

1712e63.9

,514

881

,405

1404

ba

Table 2. Preventive and/or Therapeutic Effects of ADN-138 and Withdrawal of Gelactose on the Prolongation of Peak Latencies of Oscillatory Potentials in Galactosemic Rats

ERG ABNORMALITIES AND POLYOL PATHWAY

459

prolongation of peak latencies and in some groups a reduction in the amplitude of OPs to levels similar to those in diabetic hyperglycemia. The similarities in the ERG abnormalities between diabetic and galactosemic rats suggest that diabetic hyperglycemia and galactosemia may have common mechanism(s) responsible for the development of ERG abnormalities. Insulin therapy in STZ-diabetic rats prevented and restored ERG abnormalities, in agreement with a previous report,3 and an aldose reductase inhibitor, ADN-138, also improved ERG abnormalities in both diabetic and galactosemic rats. Moreover, there was a significant correlation between the neuronal deficit and retinal hexitol accumulation. Therefore, we consider that the neuronal deficit in both hyperglycemias may result, in substantial part, from the retinal hexitol accumulation. But there is no information currently available regarding the biochemical counterparts of the neurophysiologic abnormalities in the retina. Microelectrode investigations indicate that OPs originate in the inner retinal layer in animals?3 At present, it is not known which cell type generates the oscillation, but, Heynen et al” recently reported that the most likely candidates for generation of OPs are bipolar cells. Therefore, electrophysiologic deficit may be associated with alterations of neural functions in this layer of the retina in response to hyperglycemia. On the other hand, immunohistochemical investigations have demonstrated the presence of aldose reductase in the Mtiller cells,*’ ganglion cells,25 and mural cells26 in the capillaries of the rat retina. Among the polyol-producing cells in the retina, the Miiller cells, the retinal neuroglial cells, exert a crucial metabolic function in the middle retinal layer by serving as the main nutritional and excretional transport system between the capillaries and the neural system. Therefore, functional changes in the Miiller cells possessing polyol-producing activity may be responsible for the neural dysfunctions of the inner retinal layer in hyperglycemia. Myo-inositol content of sciatic nerve has been known to be reduced in both diabetic hyperglycemia*’ and galactosemia,****!’ and it has been suggested that altered myo-inositol metabolism of sciatic nerve may be linked to nerve dysfunc-

tion in diabetic hyperglycemia.30 Furthermore, it has been demonstrated that myo-inositol content of dorsal root ganis decreased, but not kidney,33 superior glia”~‘* and lens33934 cervical ganglia,32 and retina3-’ in diabetic hyperglycemia and/or galactosemia. In this study, nerve myo-inositol content in hyperglycemic rats was certainly depleted and reversed by ADN- 138 treatment or 1% dietary myo-inositol supplements, in agreement with a previous report,*’ but it was not clear whether or not there was a significant correlation between reduced myoinositol content and nerve dysfunction (data not shown). Furthermore, retinal myo-inositol content in both diabetic and galactosemic rats was not affected, in agreement with a previously reported study.” In addition, ERG abnormalities in hyperglycemia were neither prevented nor improved by 1% dietary myo-inositol supplements (in preparation). These facts suggest that hyperglycemia doesn’t necessarily lower myo-inositol level and that hyperglycemia-associated dysfunction doesn’t always link myo-inositol metabolism in polyol-producing tissues. It has been recognized that changes in OPs are one of the most sensitive parameters of prediabetes to advanced stages Strict metabolic control with insulin therof retinopathy. ‘,2*5 apy has normalized OP abnormalities in insulin-dependent patients without retinopathy,’ and brought improvement in those with retinopathy.6 In this study, insulin and an aldose reductase inhibitor, ADN-138, had preventive and therapeutic effects on the changes of OPs in diabetic and/or galactosemic rats. These findings suggest that changes in the retina in hyperglycemic rats are reversible neurophysiologic abnormalities similar to those in human diabetes.5q6 Therefore, a new aldose reductase inhibitor, ADN-138, has potential for therapy of retinopathy. Further studies on structural changes in retinal microvasculature in advanced stages of hyperglycemia are in progress.

ACKNOWLEDGMENT The authors wish to thank N. Takahashi and H. Namiki for conscientious technical assistance. and T. Ikeda for manuscript preparation.

REFERENCES 1. Algver P: Clinical studies on the oscillatory potentials of the human electroretinogram with special reference to the scotopic b-wave. Acta Ophthalmol46:993-1024, 1968 2. Yonemura D, Aoki T, Tsuzuki K: Electroretinogram in diabetic retinopathy. Arch OphthaImol68:49-54, 1962 3. Sato S, Sugimoto S, Ando T, et al: An electrophysiological method for detecting diabetic retinopathy in rats. Nippon Yakurigaku Zasshi 84509-517, 1984 4. Tanaka K: Effects of various agents on the ERG of the streptozotocin-diabetic rat. Folia Ophthalmol Jpn 32:1470-1480, 1981 5. Frost-Larsen K, Sandahl Christiansen J, Parving H-H: The effect of strict short-term metabolic control on retinal nervous system abnormalities in newly diagnosed type I (insulin-dependent) diabetic patients. Diabetologia 24:207-209, 1983 6. Steno Study Group: Effect of 6 months of strict metabolic

control on eye and kidney function in insulin-dependent diabetics with background retinopathy. Lancet I: 121-124, 1982 7. Dvornik D, Simard-Duquesne N, Krami M, et al: Polyol accumulation in galactosemic and diabetic rates: Control by an aldose reductase inhibitor. Science 182:1146-l 148, 1973 8. Gabbay KH, Snider JJ: Nerve conduction defect in galactosefed rats. Diabetes 21:295-300, 1972 9. Engerman RL, Kern TS: Experimental galactosemia produces diabetic-like retinopathy. Diabetes 33:97-100, 1984 10. Kojima K, Matsubara H, Harada T, et al: Effects of aldose reductase inhibitor on retinal microangiopathy in streptozotocindiabetic rats. Jpn J Ophthalmol 29:99-109, 1985 11. Okuda J, Yashima K, Inagaki K, et al: Effects of an aldose reductase inhibitor, I-[(p-bromophenyl)sulfonyl]hydantoin, on cataract formation and tissue polyol levels in galactosemic rats. Chem Pharm Bull 33:2990-2995, 1985

460

12. Peterson MJ, Sarges R, Aldinger CE, et al: CP-45,634: A novel aldose reductase inhibitor that inhibits polyol pathway activity in diabetic and galactosemic rats. Metabolism 28:456-461, 1979 (suppl 1) 13. Simard-Duquesne N, Greselin E, Gonzalez R, et al: Prevention of cataract development in severely galactosemic rats by the aldose reductase inhibitor, tolrestat (42048). Proc Sot Exp Biol Med 178:605, 1985 14. Yue DK, Hanwell MA, Satchel1 PM, et al: The effect of aldose reductase inhibition on motor nerve conduction velocity in diabetic rats. Diabetes 31:789-794, 1982 15. Kikkawa R, Hatanaka I, Yasuda H, et al: Effect of a new aldose reductase inhibitor, (E)-3-carboxymethyl-5-[(2E)-methyl3-phenylpropenylidenelrhodanine (ONO-2235) on peripheral nerve disorders in streptozotocin-diabetic rats. Diabetologia 24:290-292, 1983 16. Stribling D, Mirrlees DJ, Harrison HE, et al: Properties of ICI 128,436, a novel aldose reductase inhibitor, and its effects on diabetic complications in the rat. Metabolism 34:336-344, 1985 17. Fujimori S, Hirata Y, Iinuma F, et al: Novel Spiro-linked pyrrolidine-2,5-diones as aldose reductase inhibitors. J Med Chem (in press) 18. Somogyi M: Determination of blood sugar. J Biol Chem 160:69-73, 1945 19. Sweeley CC, Bentley R, Makita M, et al: Gas-liquid chromatography of trimethylsilyl derivatives of sugars and related substances. J Am Chem Sot 85:2497-2507, 1963 20. Bergmeyer HU, Bernt E: Determination with glucose oxidase and peroxidase, in Bergmeyer HU (ed): Methods of Enzymatic Analysis, vol 3 (ed 3). San Diego, Academic, 1974, pp 1205-1215 21. Kurz G, Wallenfels K: UV-assay with galactose dehydrogenase, in Bergmeyer HU (ed): Methods of Enzymatic Analysis, vol 3 (cd 2). San Diego, Academic, 1974, pp l279- 1282 22. MacGregor LC, Matschinsky FM: Treatment with aldose reductase inhibitor or with myo-inositol arrests deterioration of the electroretinogram of diabetic rats. J Clin Invest 76:887-889, 1985

SEGAWA ET AL

23. Brown KT: The electroretinogram: Its components and their origins. Vision Res 8:633-677, 1986 24. Heynen H, Wachtmeister L, van Norren D: Origin of the oscillatory potentials in the primate retina. Vision Res 25:13651373,1985 25. Ludvigson MA, Sorenson RL: Immunohistochemical localization of aldose reductase. II. Rat eye and kidney. Diabetes 29:450-459, 1980 26. Akagi Y, Kador PF, Kuwabara T, et al: Aldose reductase localization in retinal mural cells. Invest Ophthalmol Vis Sci 24:257, 1983 27. Greene DA, De Jesus PV, Winegrad A: Effects of insulin and dietary myoinositol on impaired peripheral motor nerve conduction velocity in acute streptozotocin diabetes. J Clin Invest 55: l326- 1336, 1975 28. Stewart MA, Sherman WR, Kurien MM, et al: Polyol accumulations in nervous tissue of rats with experimental diabetes and galactosaemia. J Neurochem 14:1057-1066, 1967 29. Sharma AK, Thomas PK, Baker RWR: Peripheral nerve abnormalities related to galactose administration in rats. J Neurol Neurosurg Psychiatry 39:794-802, 1976 30. Greene DA, Lewis RA, Lattimer SA, et al: Selective effects of myo-inositol administration on sciatic and tibia1 motor nerve conduction parameters in the streptozotocin-diabetic rat. Diabetes 31:573-578, 1982 31. Green RJ, King RHM, Thomas PK, et al: Sodiumpotassium-ATPase activity in the dorsal root ganglia of rats with streptozotocin-induced diabetes. Diabetologia 28:104-107, 1985 32. Lambourne JE, Tomlinson DR. Brown AM, et al: Opposite effects of diabetes and galactosemia on adenosine triphosphatase activity in rat nervous tissue. Diabetologia 30:360-362, 1987 33. Poulson R, Heath H: Inhibition of aldose reductase in five tissues of the streptozotocin-diabetic rat. Biochem Pharmacol 32:1495-1499, 1983 34. Stewart MA, Kurien MM, Sherman WR, et al: Inositol changes in nerve and lens of galactose fed rats. J Neurochem 15:941-946, 1968