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Edema and increased endoneurial sodium in galactose neuropathy
Journal of the Neurological Sciences, 1986, 74:35-43
35
Elsevier
JNS 2658
Edema and Increased Endoneurial Sodium in Galactose Neuropathy Reversal with an Aldose Reductase Inhibitor A.P. Mizisin l, H.C. Powell 3 and R.R. Myers 1"2 ~Departments of Anesthesiology, 2Neurosciences and 3Pathology (Neuropathology), Veterans Administration Medical Center, San Diego and the University of California, San Diego, School of Medicine, V-151, La Jolla, CA 92093 (U.S.A.) (Received 16 September, 1985) (Accepted 13 January, 1986)
SUMMARY
Galactose neuropathy was produced in rats by feeding a diet containing 30~o D-galactose. After 12 weeks ofgalactose ingestion, all rats developed bilateral cataracts, polydypsia and polyuria. These galactose-intoxicated animals were divided into two groups that both continued with the galactose diet: animals that were treated with the aldose reductase inhibitor, ICI 128,436, for 4-6 weeks, and a control group of animals that received just excipient. At the end of the study, endoneurial fluid pressures, nerve water contents and endoneurial fluid electrolyte concentrations were determined from sciatic nerves of treated and untreated animals. The extent ofneuropathy in each animal was evaluated by light microscopy. Treatment of galactose-intoxicated rats with ICI 128,436 restored to normal levels the elevated endoneurial sodium concentration, increased water content and interstitial fluid pressure characteristic of galactose neuropathy. These results, obtained with an agent that blocks the sorbitol pathway, associate elevated sodium with an osmotic force contributing to edema and increased endoneurial fluid pressure in galactose neuropathy and suggest that endoneurial sodium levels are linked to blood-sugar concentration.
This work was supported by the Veterans Administration and USPHS Grants NS-14162 and NS-18715. Correspondence to: Andrew P. Mizisin, Ph.D., Anesthesia Research, V-151, University of California, San Diego, La Jolla, CA 92093, U.S.A., Tel. (619)453-7500 X3607. 0022-510X/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
36 Key words: A l d o s e reductase inhibitor - E d e m a - E l e c t r o n p r o b e m i c r o a n a l y s i s E n d o n e u r i a l electrolytes - E n d o n e u r i a l f l u i d p r e s s u r e - Galactose n e u r o p a t h y - Sorbitol p a t h w a y
INTRODUCTION Experimental galactosemic neuropathy is useful for investigating the pathophysiology of nerve edema (Myers et al. 1979; Powell et al. 1981 ; Myers and Powell 1984). Edema formation in lens and peripheral nerve results from a metabolic disorder involving the sorbitol pathway and is thought to be related to the accumulation of osmotically significant amounts of polyols within the semipermeable membranes of these tissues (for review see Kinoshita 1965; Gabbay 1973, 1975). Metabolic activity in the sorbitol pathway is dependent on two enzymes, aldose reductase and sorbitol dehydrogenase. Together with appropriate cofactors, these enzymes provide an alternate way for glucose to enter glycolytic pathways (Kador et al. 1985). Aldose reductase has a greater affinity for galactose than it does for glucose, so galactose-intoxicated animals convert more galactose to dulcitol (galactitol) than glucose to sorbitol. Since dulcitol is not oxidized further by sorbitol dehydrogenase, tissue concentrations can reach levels capable of exerting a hyperosmotic effect (Kinoshita 1965; Gabbay and Snider 1972; Sharma et al. 1976). Galactosemic animals develop a condition comparable in many respects to diabetes mellitus (for review see Kador et al. 1985). In galactosemic animals, endoneurial edema is one of the earliest structural abnormalities to become apparent (Myers et al. 1979). After 12 weeks ofgalactose ingestion, increased endoneurial edema produces a significant elevation in endoneurial fluid pressure (Myers et al. 1979). Other complications in the nerves of galactosemic animals include reductions in nerve myoinositol content, axonal transport and conduction velocity (Stewart et al. 1967; Gabbay and Snider 1972; Sharma et al. 1976; Tomlinson et al. 1982). The decrease in nerve conduction velocity is an early phenomenon and apparently precedes the decrease in nerve blood flow that occurs later in the neuropathy during the interval between the onset of elevated endoneurial fluid pressure and the appearance of nerve fiber damage (Low et al. 1982; Myers and Powell 1984). In advanced stages of galactose neuropathy, there is extensive primary demyelination associated with changes in Schwann cell morphology, as well as axonal degeneration (Powell et al. 1981; Powell and Myers 1983; Myers and Powell 1984). Increased interstitial sodium concentrations averaging 295 mEq/1 have recently been measured in the endoneurial fluid of galactosemic animals receiving a diet containing 40~ galactose (Mizisin et al. 1986). This value is nearly twice the sodium concentration in normal rats, suggesting that elevated endoneurial sodium contributes to the osmotic force causing edema and increased endoneurial fluid pressure in this neuropathy. These endoneurial electrolyte changes, in conjunction with the fact that the high blood-sugar levels maintained by galactose intoxication provide a concentration gradient that favors the movement of sugar into the endoneurium, further suggested to
37 us that endoneurial sodium accumulation might be linked to the movement of sugar into the endoneurium. In essence, we hypothesize that the sorbitol pathway contributes to the pathophysiologic changes in galactose neuropathy by maintaining an inward sugar concentration gradient that promotes the accumulation of osmotically significant amounts of sodium in the endoneurial interstitium. Therefore, if elevated sodium concentrations are associated with the movement of sugar across the blood-nerve barrier and with edema and increased interstitial fluid pressure, then chemical agents that block the sorbitol pathway should ameliorate these conditions. The work described below demonstrates that treatment of galactose-intoxicated rats with the aldose reductase inhibitor, ICI 128,436, restores peripheral nerve water content, interstitial fluid pressure, and endoneurifl sodium concentrations to normal levels.
MATERIALS AND METHODS
Twenty one, age-matched, female Sprague-Dawley rats housed in cages with wire bottoms were fed a 30~ D-galactose diet for 16-18 weeks. The galactose was provided in biscuits made with powdered rat chow, D-galactose (30 ~o of dry ingredients by weight) and water mixed together and oven-dried. After 12 weeks 15 rats received the aldose reductase inhibitor, ICI 128,436, orally by gavage once daily for 4-6 weeks while continuing to receive 30~o galactose biscuits and water ad libitum. ICI 128,436 was suspended by ball-milling in 0.5Yo Tween 80 and given at a dose of 25 mg/kg/day (Stribling et al. 1985). An additional 6 galactose-intoxicated rats served as controls and received a daily gavage of just excipient for 4-6 weeks while continuing on the galactose diet. After 16-18 weeks the rats were anesthetized with a mixture (2 ml/kg) of pentobarbital (50 mg/ml), diazepam (5 mg/ml) and saline in volume proportions of 1 : 1 : 2 given intraperitoneally. The sciatic nerves were then exposed and used in determinations of endoneurial fluid pressure (EFP), nerve water content and endoneurial fluid (EF) electrolyte concentrations. Thick sections taken from one or both of the sciatic nerves of each animal were examined by light microscopy for the presence of edema. One sciatic nerve was routinely used for either EFP or EF electrolyte measurements and histology, and the contralateral nerve for microgravimetric determination of nerve water content. These techniques are described below. EFP
Endoneurial fluid pressures were recorded using the servo-null micropipette method described in detail elsewhere (Myers et al. 1978). In short, this method is based on changes induced in the electrical characteristics of the tip of a fluid-filled glass micropipette when the tip is inserted through the perineurium into the endoneurial fluid space of an exposed sciatic nerve fascicle. Positive endoneurial fluid pressures in the endoneurium force interstitial fluid into the micropipette tip, thereby altering its resistivity and creating an error signal. The error signal, sensed by a Wheatstone bridge, drives a linear motor that forces a reservoir of micropipette fluid back down the pipette
38 shank until the error signal is minimized. The hydraulic force required to minimize the error signal is considered equivalent to the EFP of the nerve fascicle. Nerve water content
The water content of sciatic nerve was determined using the method of microgravimetric analysis described by Costello et al. (1982). Briefly, a density gradient column is prepared from two nonaqueous fluids with high and low specific gravities. The relationship between depth in the column and specific gravity is calibrated using droplets of salt solutions with known specific gravity. The gradient position of nerve in the density gradient column is then related to its specific gravity which is in turn correlated to the percentage of water in the tissue. E F electrolytes
The analysis of microdrop samples of EF has been described previously (Myers et al. 1983). In essence, endoneurial fluid is collected with a teflon-coated, glass micropipette after a tiny incision is made in the perineurium of a nerve fascicle. The aspirated EF is immediately transferred to a petri dish containing water-saturated hexadecane to prevent volume loss due to evaporation. A teflon-coated picoliter pipette is then used to transfer equal volumes of EF and control fluids (serum and standard solutions) to a parlodion film supported by a nickel grid. After flash-evaporation under vacuum, microanalysis of the microdrops is performed with a scanning electron microscope (Etec Autoscan) equipped with an x-ray spectrometer (Kevex 100). Histology
Sciatic nerves were processed for light microscopy by fixing in 2.5 ~o phosphatebuffered glutaraldehyde for 24 h, postfbting in osmium tetroxide for 2 h, dehydrating and embedding in araldite. One-/am-thick sections were stained with paraphenylenediamine. The presence and extent of edema were assessed semiquantitatively by assigning an edema score ranging from 0 to 4 in 0.5 step increments. A score of 0 indicated no edema. Extensive edema in the subperineurial space (1 point), in perivascular spaces (lpoint), along intrafascicular endoneurial membrane partitions (1 point), and in interstitial spaces between individual axons (1 point) was assigned a score of 4. 0.5 point increments were used to discriminate between differences in edema within any single region of nerve. Statistics
The data were analyzed with the Student's t-test except for the histologic assessment of edema in which the difference between experimental groups was assessed with the Mann-Whitney U-test (Zar 1984). RESULTS
After 12 weeks on a diet containing 30~o D-galactose, all rats in this study developed bilateral cataracts, polydypsia and polyuria. Endoneurial fluid pressure of
39 TABLE 1 THE EFFECT OF THE ALDOSE REDUCTASE INHIBITOR, ICI 128,436, ON THE ELEVATED ENDONEURIAL FLUID PRESSURE AND WATER CONTENT OF PERIPHERAL NERVE ASSOCIATED WITH GALACTOSE NEUROPATHY Sample size is indicated in parentheses. Data are expressed as mean _+standard deviation. Differences are significant (p < 0.001; two-tailed Student's t-test).
EFP (cm H20) Water content (%)
Untreated
Treated
8.1 + 0.9 (2) 76.0 + 4.2 (4)
2.2 _+0.6 (6) 66.3 + 2.0 (11)
galactose-intoxicated rats in this study was 4 t i m e s that r e c o r d e d in n o r m a l S p r a g u e - D a w l e y rats (Table 1 ; 2.0 c m H 2 0 , M y e r s et al. 1979). T r e a t m e n t o f g a l a c t o s e intoxicated rats with the aldose reductase inhibitor, I C I 128,436, restored E F P to n o r m a l levels (Table 1). The nerve water content o f sciatic nerves o f untreated galactoseintoxicated rats was 10~o greater than that o f age-matched, galactose-intoxicated rats treated with I C I 128,436 (Table 1); the latter was c o m p a r a b l e to the nerve water content o f n o r m a l Sprague--Dawley rats ( 6 6 ~ , M y e r s et al. 1984). T r e a t m e n t with the aldose reductase inhibitor, I C I 128,436, was associated with a significant reduction in endoneurial fluid concentrations o f sodium and chloride ions c o m p a r e d to untreated galactose-intoxicated rats (Table 2). Sodium and chloride ion concentration in the endoneurial interstitium o f treated rats a p p r o a c h e d the electrolyte levels o f n o r m a l S p r a g n e - D a w l e y rats (sodium = 152.4 mEq/1, chloride = 122.2 mEq/l, Mizisin et al. 1986). Serum levels o f sodium, chloride a n d p o t a s s i u m ions o f treated and u n t r e a t e d galactose-intoxicated rats (mean + s t a n d a r d deviation) were n o t significantly different (treated: s o d i u m = 142.8 + 1.5 mEq/l, chloride = 107.2 + 2.8 mEq/l, p o t a s -
TABLE 2 THE EFFECT OF THE ALDOSE REDUCTASE INHIBITOR, ICI 128,436, ON ENDONEURIAL FLUID ELECTROLYTE CONCENTRATIONS (mEq/1) OF SCIATIC NERVE FROM GALACTOSE-INTOXICATED SPRAGUE-DAWLEY RATS Electrolyte measurements were made on 5 untreated and 5 treated galactose-intoxicated rats. Data are expressed as mean + standard deviation and were analyzed with two-tailed Student's t-test.
Sodium Chloride Potassium * P < 0.001.
** P < 0.02.
Untreated
Treated
234.0 _+22.6* 156.0 + 11.2"* 12.6 _+ 5.7
171.7 + 14.4" 105.6 + 32.7** 9.9 +_ 5.8
40 TABLE 3 T H E E F F E C T OF T R E A T M E N T W I T H T H E A L D O S E R E D U C T A S E INHIBITOR, ICI 128,436, ON P E R I P H E R A L NERVE E D E M A A S S O C I A T E D W I T H G A L A C T O S E N E U R O P A T H Y . Edema was evaluated semiquantitatively using a scale ranging from 0 (no edema) to 4 (extensive edema in the subperineurial, interstitial and perivascular regions of the endoneurium) in 0.5 step increments. Data are presented as mean + standard deviation. Difference is significant (P < 0.001; M a n n - W h i t n e y U-test).
Edema score ( 0 - 4 )
Untreated a
Treated u
2.9 +_ 1.1
0.2 _+ 0.3
a 7 nerves from 6 animals. b 14 nerves from 12 animals.
sium = 4.2 + 0.7 mEq/1; untreated: sodium = 144.6 + 1.1 mEq/1, chloride = 108.4 + 2.5 mEq/1, p o t a s s i u m = 4.6 + 1.1 m E q / l ) and were c o m p a r a b l e to serum levels of n o r m a l S p r a g u e - D a w l e y rats (sodium = 141.5 mEq/l, chloride = 102.7 mEq/l, p o t a s sium = 7.8 mEq/l, Mizisin et al. 1986).
Fig. 1. Sciatic nerve from galactose-intoxicated rats. A : Rats fed a diet containing 30 % D-galactose; B: Rats fed a 30% D-galactose diet and treated with the aldose reductase inhibitor, ICI 128,436. Paraphenylenediamine, original magnification × 250.
41 Peripheral nerve edema associated with galactose neuropathy was ameliorated by treatment with the aldose reductase inhibitor, ICI 128,436 (Table 3 and Fig. 1). In most instances, transverse sections of sciatic nerve from treated, galactose-intoxicated rats lacked the subperineurial, interstitial and perivascular edema characteristic of sciatic nerve from untreated, galactose-intoxicated rats. DISCUSSION Treatment of galactose-intoxicated rats with the aldose reductase inhibitor, ICI 128,436, reverses the increases in endoneurial sodium concentration, water content and interstitial fluid pressure that characterize the early stages of galactose neuropathy (Tables 1, 2 and 3; Fig. 1). In experimental models of diabetes, aldose reductase inhibitors have been shown to improve nerve function (Yue et al. 1982; Kikkawa et al. 1983) and axonal transport (Tomlinson et al. 1982), and to reverse increases in nerve water content (Robison 1984). In humans, aldose reductase inhibitors have been reported to improve nerve conduction velocity deficits associated with diabetic neuropathy (Handelsman and Turtle 1981; Jaspan et al. 1983; Judzewitsch et al. 1983; Young et al. 1983). Therapeutic amelioration of edema and concomitant reduction of elevated endoneurial sodium concentration demonstrated here suggests that endoneurial sodium contributes to the osmotic force generating edema and increased endoneurial fluid pressure in galactose neuropathy. Further support for sodium being the principal osmolyte responsible for edema in galactose neuropathy is provided by a comparison of the magnitude of the osmotic contribution of polyhydric sugars from sciatic nerve of galactose-intoxicated rats (about 18 mOsm/l on 40~o galactose diet, Sharma et al. 1976) to that of endoneurial sodium (140 mOsm/1 on 40~/o galactose diet, Mizisin et al. 1986). The covariance of endoneurial sodium concentration, edema and interstitial fluid pressure in response to inhibition of a key enzyme of the sorbitol pathway is consistent with our suggestion that sodium concentrations are linked to the amount of sugar available for movement into the endoneurium of peripheral nerve. Further support comes from our preliminary observation that endoneurial sodium appears to be related, in a dose-dependent manner, to the amount of galactose provided in the diet. In 6 rats fed a diet containing 40~o galactose, endoneurial sodium averaged 295 mEq/1 (Mizisin et ai. 1986). In the 5 untreated rats fed a 30~o galactose diet in this study, endoneurial sodium concentration averaged 234 mEq/l (Table 2). The endoneurial sodium concentration of normal rats is 152 mEq/1 (Mizisin et al. 1986). It is conceivable that endoneurial sodium accumulation is dependent on the movement of sugar into the endoneurium so that the eventual sodium concentration is related to the sugar-concentration gradient across the blood-nerve barrier. While the mechanism responsible for enhanced hypertonicity of sodium is not known, it may be related to the movement of sugar into the endoneurial compartment. In other insulinindependent tissues such as intestine (Frizzel et al. 1973) and proximal kidney tubule (Ullrich 1976) co-migration of sodium and glucose and/or galactose has been demonstrated. A sugar-concentration gradient would be maintained by endoneurial metabolic
42 p r o c e s s e s t h a t use g l u c o s e a n d / o r g a l a c t o s e (i.e. glycolysis, p o l y o l synthesis via the sorbitol p a t h w a y ,
nonenzymatic
glycosylation, glycogenesis).
Participation
o f the
sorbitol p a t h w a y in the m a i n t e n a n c e o f this s u g a r - c o n c e n t r a t i o n g r a d i e n t c o u l d explain h o w inhibition o f a l d o s e r e d u c t a s e results in d e c r e a s e d e n d o n e u r i a l s o d i u m levels in galactose neuropathy. ACKNOWLEDGEMENTS T h e a u t h o r s gratefully a c k n o w l e d g e t h e e x p e r t t e c h n i c a l a s s i s t a n c e o f M s . H e i d i M. H e c k m a n . T h e a l d o s e r e d u c t a s e inhibitor, I C I 128,436, w a s kindly s u p p l i e d by I C I , Pharmaceutical Division, U.S.A.
REFERENCES Costello, M.L., H.C. Powell and R.R. Myers (1982) Microgravimetry of nerve edema, Muscle and Nerve, 5: 261-264. Frizzel, R.A., H.N. Nellans and S.G. Schultz (1973) Effects of sugars and amino acids on sodium and potassium influx in the rabbit ileum, J. Clin. Invest., 52: 215-217. Gabbay, K. H. (1973) The sorbitol pathway and complications of diabetes, N. Engl. J. Med., 288: 831-836. Gabbay, K. H. (1975) Hyperglycemia, polyol metabolism, and complications of diabetes mellitus, Ann. Rev. Med., 26: 521-536. Gabbay, K. H. and J.J. Snider (1972) Nerve conduction defect in galactose-fed rats, Diabetes, 21 : 295-300. Handelsman, D.J. and J.R. Turtle (1981) Clinical trial of an aldose reductase inhibitor in diabetic neuropathy, Diabetes, 30: 459-464. Jaspan, J., R. MaseUi, K. Herold and C. Bartkus (1983) Treatment of severely painful diabetic neuropathy with an aldose reductase inhibitor - - Relief of pain and improved somatic and autonomic nerve function, Lancet, ii: 758-762. Judzewitsch, R.G., J.B. Jaspan, K.S. Polonsky, C.R. Weinberg, J.B. Halter, E. Halar, M.A. Pfeifer, C. Vukadinovic, L. Bernstein, M. Schneider, Kung-Yee Liang, K.H. Gabbay, A.H. Rubenstein and D. Porte (1983) Aldose reduetase inhibition improves nerve conduction velocity in diabetic patients, N. Engl. J. Med., 308: 119-125. Kador, P.F., W. G. Robison and J. H. Kinoshita (1985) The pharmacology of aldose reductase inhibitors, Ann. Rev. Pharmacol. Toxicol., 25: 691-714. Kikkawa, R., I. Hatanaka, H. Yasuda, N. Kobayashi, Y. Shigeta, H. Terashima, T. Morimura and M. Tsuboshima (1983) Effect of a new aldose reductase inhibitor, (E)-3-carboxymethyl-5-((2E)-methyl3-phenylpropenylidene) rhodanine (ONO-2235) on peripheral nerve disorders in streptozotocindiabetic rats, Diabetol., 24: 290-292. Kinoshita, J.H. (1965) Cataracts in galactosemia - - The Jonas Friedenwald Memorial Lecture, Invest. Ophthalmol., 4: 786-799. Kung-Yee Liang, K. H. Gabbay, A. H. Rubenstein and D. Porte (1983) Aldose reductase inhibition improves nerve conduction velocity in diabetic patients, N. Engl. J. Med., 308:119-125. Low, P.A., P.J. Dyck and J.D. Schmelzer (1982) Chronic elevation of endoneurial pressure is associated with low-grade pathology, Muscle and Nerve, 5: 162-165. Mizisin, A.P., R.R. Myers and H.C. PoweU (1986) Endoneurial sodium accumulation in galactosemic rat nerves, Muscle and Nerve, 9: 440-444. Myers, R.R. and H.C. PoweU (1984) Galactose neuropathy - - Impact of chronic endoneurial edema on nerve blood flow, Ann. Neurol., 16: 587-594. Myers, R.R., H.C. Powell, M.C. Costelio, P.W. Lampert and B.W. Zweifach (1978) Endoneurial fluid pressure - - Direct measurement with micropipettes, Brain Res., 148: 510-515. Myers, R.R., M.C. Costello and H.C. PoweU (1979) Increased endoneurial fluid pressure in galactose neuropathy, Muscle and Nerve, 2: 299-303. Myers, R.R., H.M. Heekman and H.C. Powell (1983) Endoneurial fluid is hypertonic, J. Neuropath. Exp. Neurol., 42: 217-224.
43 Powell, H.C. and R.R. Myers (1983) Schwann cell changes and demyelination in chronic galactose neuropathy, Muscle and Nerve, 6: 218-227. Powell, H.C., M. L. Costello and R.R. Myers (1981) Endoneurial fluid pressure in experimental models of diabetic neuropathy, J. Neuropath. Exp. Neurol., 40: 613-624. Robison, W. G. (1984) Aldose reductase and diabetic neuropathy. In: D. G. Cogan (mod.), Aldose reductase and complications of diabetes, Ann. Intern. Med., 101: 82-91. Sharma, A. K., P. K. Thomas and R. W. R. Baker (1976) Peripheral nerve abnormalities related to galactose administration in rats, J. Neurol. Neurosurg. Psychiat., 39: 794-802. Steward, M.A., W.R. Sherman, M.M. Kurien, G.I. Moonsammy and M. Wisgerhof (1967) Polyol accumulation in nervous tissue of rats with experimental diabetes and galactosemia, J. Neurochem., 14: 1057-1066. Stribling, D., D.J. Mirrless, H.E. Harrison and D. C. N. Earl (1985) Properties oflCI 128,436: a novel aldose reductase inhibitor and its effects on diabetic complications in the rat, Metabolism, 34: 336-344. Tomlinson, D.R., P.R. Holmes and J.H. Mayer (1982) Reversal, by treatment with an aldose reductase inhibitor, of impaired axonal transport and motor nerve conduction velocity in experimental diabetes mellitus, Neurosci. Lett., 31 : 189-193. Ullrich, K.N. (1976) Renal tubular mechanisms of organic solute transport, Kidney Int., 9:134-148. Young, R.J., D.J. Ewing and B. F. Clarke (1983) A controlled trial of sorbinil, an aldose reductase inhibitor, in chronic painful diabetic neuropathy, Diabetes, 32: 938-942. Yue, D.K., M.A. Hanwell, P.M. Satchell and J.R. Turtle (1982) The effect of aldose reductase inhibition on motor nerve conduction velocity in diabetic rats, Diabetes, 31: 789-794. Zar, J.H. (1984) Biostatistical Analysis, Prentice-Hall, Inc., Englewood Cliffs, NJ.
35
Elsevier
JNS 2658
Edema and Increased Endoneurial Sodium in Galactose Neuropathy Reversal with an Aldose Reductase Inhibitor A.P. Mizisin l, H.C. Powell 3 and R.R. Myers 1"2 ~Departments of Anesthesiology, 2Neurosciences and 3Pathology (Neuropathology), Veterans Administration Medical Center, San Diego and the University of California, San Diego, School of Medicine, V-151, La Jolla, CA 92093 (U.S.A.) (Received 16 September, 1985) (Accepted 13 January, 1986)
SUMMARY
Galactose neuropathy was produced in rats by feeding a diet containing 30~o D-galactose. After 12 weeks ofgalactose ingestion, all rats developed bilateral cataracts, polydypsia and polyuria. These galactose-intoxicated animals were divided into two groups that both continued with the galactose diet: animals that were treated with the aldose reductase inhibitor, ICI 128,436, for 4-6 weeks, and a control group of animals that received just excipient. At the end of the study, endoneurial fluid pressures, nerve water contents and endoneurial fluid electrolyte concentrations were determined from sciatic nerves of treated and untreated animals. The extent ofneuropathy in each animal was evaluated by light microscopy. Treatment of galactose-intoxicated rats with ICI 128,436 restored to normal levels the elevated endoneurial sodium concentration, increased water content and interstitial fluid pressure characteristic of galactose neuropathy. These results, obtained with an agent that blocks the sorbitol pathway, associate elevated sodium with an osmotic force contributing to edema and increased endoneurial fluid pressure in galactose neuropathy and suggest that endoneurial sodium levels are linked to blood-sugar concentration.
This work was supported by the Veterans Administration and USPHS Grants NS-14162 and NS-18715. Correspondence to: Andrew P. Mizisin, Ph.D., Anesthesia Research, V-151, University of California, San Diego, La Jolla, CA 92093, U.S.A., Tel. (619)453-7500 X3607. 0022-510X/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
36 Key words: A l d o s e reductase inhibitor - E d e m a - E l e c t r o n p r o b e m i c r o a n a l y s i s E n d o n e u r i a l electrolytes - E n d o n e u r i a l f l u i d p r e s s u r e - Galactose n e u r o p a t h y - Sorbitol p a t h w a y
INTRODUCTION Experimental galactosemic neuropathy is useful for investigating the pathophysiology of nerve edema (Myers et al. 1979; Powell et al. 1981 ; Myers and Powell 1984). Edema formation in lens and peripheral nerve results from a metabolic disorder involving the sorbitol pathway and is thought to be related to the accumulation of osmotically significant amounts of polyols within the semipermeable membranes of these tissues (for review see Kinoshita 1965; Gabbay 1973, 1975). Metabolic activity in the sorbitol pathway is dependent on two enzymes, aldose reductase and sorbitol dehydrogenase. Together with appropriate cofactors, these enzymes provide an alternate way for glucose to enter glycolytic pathways (Kador et al. 1985). Aldose reductase has a greater affinity for galactose than it does for glucose, so galactose-intoxicated animals convert more galactose to dulcitol (galactitol) than glucose to sorbitol. Since dulcitol is not oxidized further by sorbitol dehydrogenase, tissue concentrations can reach levels capable of exerting a hyperosmotic effect (Kinoshita 1965; Gabbay and Snider 1972; Sharma et al. 1976). Galactosemic animals develop a condition comparable in many respects to diabetes mellitus (for review see Kador et al. 1985). In galactosemic animals, endoneurial edema is one of the earliest structural abnormalities to become apparent (Myers et al. 1979). After 12 weeks ofgalactose ingestion, increased endoneurial edema produces a significant elevation in endoneurial fluid pressure (Myers et al. 1979). Other complications in the nerves of galactosemic animals include reductions in nerve myoinositol content, axonal transport and conduction velocity (Stewart et al. 1967; Gabbay and Snider 1972; Sharma et al. 1976; Tomlinson et al. 1982). The decrease in nerve conduction velocity is an early phenomenon and apparently precedes the decrease in nerve blood flow that occurs later in the neuropathy during the interval between the onset of elevated endoneurial fluid pressure and the appearance of nerve fiber damage (Low et al. 1982; Myers and Powell 1984). In advanced stages of galactose neuropathy, there is extensive primary demyelination associated with changes in Schwann cell morphology, as well as axonal degeneration (Powell et al. 1981; Powell and Myers 1983; Myers and Powell 1984). Increased interstitial sodium concentrations averaging 295 mEq/1 have recently been measured in the endoneurial fluid of galactosemic animals receiving a diet containing 40~ galactose (Mizisin et al. 1986). This value is nearly twice the sodium concentration in normal rats, suggesting that elevated endoneurial sodium contributes to the osmotic force causing edema and increased endoneurial fluid pressure in this neuropathy. These endoneurial electrolyte changes, in conjunction with the fact that the high blood-sugar levels maintained by galactose intoxication provide a concentration gradient that favors the movement of sugar into the endoneurium, further suggested to
37 us that endoneurial sodium accumulation might be linked to the movement of sugar into the endoneurium. In essence, we hypothesize that the sorbitol pathway contributes to the pathophysiologic changes in galactose neuropathy by maintaining an inward sugar concentration gradient that promotes the accumulation of osmotically significant amounts of sodium in the endoneurial interstitium. Therefore, if elevated sodium concentrations are associated with the movement of sugar across the blood-nerve barrier and with edema and increased interstitial fluid pressure, then chemical agents that block the sorbitol pathway should ameliorate these conditions. The work described below demonstrates that treatment of galactose-intoxicated rats with the aldose reductase inhibitor, ICI 128,436, restores peripheral nerve water content, interstitial fluid pressure, and endoneurifl sodium concentrations to normal levels.
MATERIALS AND METHODS
Twenty one, age-matched, female Sprague-Dawley rats housed in cages with wire bottoms were fed a 30~ D-galactose diet for 16-18 weeks. The galactose was provided in biscuits made with powdered rat chow, D-galactose (30 ~o of dry ingredients by weight) and water mixed together and oven-dried. After 12 weeks 15 rats received the aldose reductase inhibitor, ICI 128,436, orally by gavage once daily for 4-6 weeks while continuing to receive 30~o galactose biscuits and water ad libitum. ICI 128,436 was suspended by ball-milling in 0.5Yo Tween 80 and given at a dose of 25 mg/kg/day (Stribling et al. 1985). An additional 6 galactose-intoxicated rats served as controls and received a daily gavage of just excipient for 4-6 weeks while continuing on the galactose diet. After 16-18 weeks the rats were anesthetized with a mixture (2 ml/kg) of pentobarbital (50 mg/ml), diazepam (5 mg/ml) and saline in volume proportions of 1 : 1 : 2 given intraperitoneally. The sciatic nerves were then exposed and used in determinations of endoneurial fluid pressure (EFP), nerve water content and endoneurial fluid (EF) electrolyte concentrations. Thick sections taken from one or both of the sciatic nerves of each animal were examined by light microscopy for the presence of edema. One sciatic nerve was routinely used for either EFP or EF electrolyte measurements and histology, and the contralateral nerve for microgravimetric determination of nerve water content. These techniques are described below. EFP
Endoneurial fluid pressures were recorded using the servo-null micropipette method described in detail elsewhere (Myers et al. 1978). In short, this method is based on changes induced in the electrical characteristics of the tip of a fluid-filled glass micropipette when the tip is inserted through the perineurium into the endoneurial fluid space of an exposed sciatic nerve fascicle. Positive endoneurial fluid pressures in the endoneurium force interstitial fluid into the micropipette tip, thereby altering its resistivity and creating an error signal. The error signal, sensed by a Wheatstone bridge, drives a linear motor that forces a reservoir of micropipette fluid back down the pipette
38 shank until the error signal is minimized. The hydraulic force required to minimize the error signal is considered equivalent to the EFP of the nerve fascicle. Nerve water content
The water content of sciatic nerve was determined using the method of microgravimetric analysis described by Costello et al. (1982). Briefly, a density gradient column is prepared from two nonaqueous fluids with high and low specific gravities. The relationship between depth in the column and specific gravity is calibrated using droplets of salt solutions with known specific gravity. The gradient position of nerve in the density gradient column is then related to its specific gravity which is in turn correlated to the percentage of water in the tissue. E F electrolytes
The analysis of microdrop samples of EF has been described previously (Myers et al. 1983). In essence, endoneurial fluid is collected with a teflon-coated, glass micropipette after a tiny incision is made in the perineurium of a nerve fascicle. The aspirated EF is immediately transferred to a petri dish containing water-saturated hexadecane to prevent volume loss due to evaporation. A teflon-coated picoliter pipette is then used to transfer equal volumes of EF and control fluids (serum and standard solutions) to a parlodion film supported by a nickel grid. After flash-evaporation under vacuum, microanalysis of the microdrops is performed with a scanning electron microscope (Etec Autoscan) equipped with an x-ray spectrometer (Kevex 100). Histology
Sciatic nerves were processed for light microscopy by fixing in 2.5 ~o phosphatebuffered glutaraldehyde for 24 h, postfbting in osmium tetroxide for 2 h, dehydrating and embedding in araldite. One-/am-thick sections were stained with paraphenylenediamine. The presence and extent of edema were assessed semiquantitatively by assigning an edema score ranging from 0 to 4 in 0.5 step increments. A score of 0 indicated no edema. Extensive edema in the subperineurial space (1 point), in perivascular spaces (lpoint), along intrafascicular endoneurial membrane partitions (1 point), and in interstitial spaces between individual axons (1 point) was assigned a score of 4. 0.5 point increments were used to discriminate between differences in edema within any single region of nerve. Statistics
The data were analyzed with the Student's t-test except for the histologic assessment of edema in which the difference between experimental groups was assessed with the Mann-Whitney U-test (Zar 1984). RESULTS
After 12 weeks on a diet containing 30~o D-galactose, all rats in this study developed bilateral cataracts, polydypsia and polyuria. Endoneurial fluid pressure of
39 TABLE 1 THE EFFECT OF THE ALDOSE REDUCTASE INHIBITOR, ICI 128,436, ON THE ELEVATED ENDONEURIAL FLUID PRESSURE AND WATER CONTENT OF PERIPHERAL NERVE ASSOCIATED WITH GALACTOSE NEUROPATHY Sample size is indicated in parentheses. Data are expressed as mean _+standard deviation. Differences are significant (p < 0.001; two-tailed Student's t-test).
EFP (cm H20) Water content (%)
Untreated
Treated
8.1 + 0.9 (2) 76.0 + 4.2 (4)
2.2 _+0.6 (6) 66.3 + 2.0 (11)
galactose-intoxicated rats in this study was 4 t i m e s that r e c o r d e d in n o r m a l S p r a g u e - D a w l e y rats (Table 1 ; 2.0 c m H 2 0 , M y e r s et al. 1979). T r e a t m e n t o f g a l a c t o s e intoxicated rats with the aldose reductase inhibitor, I C I 128,436, restored E F P to n o r m a l levels (Table 1). The nerve water content o f sciatic nerves o f untreated galactoseintoxicated rats was 10~o greater than that o f age-matched, galactose-intoxicated rats treated with I C I 128,436 (Table 1); the latter was c o m p a r a b l e to the nerve water content o f n o r m a l Sprague--Dawley rats ( 6 6 ~ , M y e r s et al. 1984). T r e a t m e n t with the aldose reductase inhibitor, I C I 128,436, was associated with a significant reduction in endoneurial fluid concentrations o f sodium and chloride ions c o m p a r e d to untreated galactose-intoxicated rats (Table 2). Sodium and chloride ion concentration in the endoneurial interstitium o f treated rats a p p r o a c h e d the electrolyte levels o f n o r m a l S p r a g n e - D a w l e y rats (sodium = 152.4 mEq/1, chloride = 122.2 mEq/l, Mizisin et al. 1986). Serum levels o f sodium, chloride a n d p o t a s s i u m ions o f treated and u n t r e a t e d galactose-intoxicated rats (mean + s t a n d a r d deviation) were n o t significantly different (treated: s o d i u m = 142.8 + 1.5 mEq/l, chloride = 107.2 + 2.8 mEq/l, p o t a s -
TABLE 2 THE EFFECT OF THE ALDOSE REDUCTASE INHIBITOR, ICI 128,436, ON ENDONEURIAL FLUID ELECTROLYTE CONCENTRATIONS (mEq/1) OF SCIATIC NERVE FROM GALACTOSE-INTOXICATED SPRAGUE-DAWLEY RATS Electrolyte measurements were made on 5 untreated and 5 treated galactose-intoxicated rats. Data are expressed as mean + standard deviation and were analyzed with two-tailed Student's t-test.
Sodium Chloride Potassium * P < 0.001.
** P < 0.02.
Untreated
Treated
234.0 _+22.6* 156.0 + 11.2"* 12.6 _+ 5.7
171.7 + 14.4" 105.6 + 32.7** 9.9 +_ 5.8
40 TABLE 3 T H E E F F E C T OF T R E A T M E N T W I T H T H E A L D O S E R E D U C T A S E INHIBITOR, ICI 128,436, ON P E R I P H E R A L NERVE E D E M A A S S O C I A T E D W I T H G A L A C T O S E N E U R O P A T H Y . Edema was evaluated semiquantitatively using a scale ranging from 0 (no edema) to 4 (extensive edema in the subperineurial, interstitial and perivascular regions of the endoneurium) in 0.5 step increments. Data are presented as mean + standard deviation. Difference is significant (P < 0.001; M a n n - W h i t n e y U-test).
Edema score ( 0 - 4 )
Untreated a
Treated u
2.9 +_ 1.1
0.2 _+ 0.3
a 7 nerves from 6 animals. b 14 nerves from 12 animals.
sium = 4.2 + 0.7 mEq/1; untreated: sodium = 144.6 + 1.1 mEq/1, chloride = 108.4 + 2.5 mEq/1, p o t a s s i u m = 4.6 + 1.1 m E q / l ) and were c o m p a r a b l e to serum levels of n o r m a l S p r a g u e - D a w l e y rats (sodium = 141.5 mEq/l, chloride = 102.7 mEq/l, p o t a s sium = 7.8 mEq/l, Mizisin et al. 1986).
Fig. 1. Sciatic nerve from galactose-intoxicated rats. A : Rats fed a diet containing 30 % D-galactose; B: Rats fed a 30% D-galactose diet and treated with the aldose reductase inhibitor, ICI 128,436. Paraphenylenediamine, original magnification × 250.
41 Peripheral nerve edema associated with galactose neuropathy was ameliorated by treatment with the aldose reductase inhibitor, ICI 128,436 (Table 3 and Fig. 1). In most instances, transverse sections of sciatic nerve from treated, galactose-intoxicated rats lacked the subperineurial, interstitial and perivascular edema characteristic of sciatic nerve from untreated, galactose-intoxicated rats. DISCUSSION Treatment of galactose-intoxicated rats with the aldose reductase inhibitor, ICI 128,436, reverses the increases in endoneurial sodium concentration, water content and interstitial fluid pressure that characterize the early stages of galactose neuropathy (Tables 1, 2 and 3; Fig. 1). In experimental models of diabetes, aldose reductase inhibitors have been shown to improve nerve function (Yue et al. 1982; Kikkawa et al. 1983) and axonal transport (Tomlinson et al. 1982), and to reverse increases in nerve water content (Robison 1984). In humans, aldose reductase inhibitors have been reported to improve nerve conduction velocity deficits associated with diabetic neuropathy (Handelsman and Turtle 1981; Jaspan et al. 1983; Judzewitsch et al. 1983; Young et al. 1983). Therapeutic amelioration of edema and concomitant reduction of elevated endoneurial sodium concentration demonstrated here suggests that endoneurial sodium contributes to the osmotic force generating edema and increased endoneurial fluid pressure in galactose neuropathy. Further support for sodium being the principal osmolyte responsible for edema in galactose neuropathy is provided by a comparison of the magnitude of the osmotic contribution of polyhydric sugars from sciatic nerve of galactose-intoxicated rats (about 18 mOsm/l on 40~o galactose diet, Sharma et al. 1976) to that of endoneurial sodium (140 mOsm/1 on 40~/o galactose diet, Mizisin et al. 1986). The covariance of endoneurial sodium concentration, edema and interstitial fluid pressure in response to inhibition of a key enzyme of the sorbitol pathway is consistent with our suggestion that sodium concentrations are linked to the amount of sugar available for movement into the endoneurium of peripheral nerve. Further support comes from our preliminary observation that endoneurial sodium appears to be related, in a dose-dependent manner, to the amount of galactose provided in the diet. In 6 rats fed a diet containing 40~o galactose, endoneurial sodium averaged 295 mEq/1 (Mizisin et ai. 1986). In the 5 untreated rats fed a 30~o galactose diet in this study, endoneurial sodium concentration averaged 234 mEq/l (Table 2). The endoneurial sodium concentration of normal rats is 152 mEq/1 (Mizisin et al. 1986). It is conceivable that endoneurial sodium accumulation is dependent on the movement of sugar into the endoneurium so that the eventual sodium concentration is related to the sugar-concentration gradient across the blood-nerve barrier. While the mechanism responsible for enhanced hypertonicity of sodium is not known, it may be related to the movement of sugar into the endoneurial compartment. In other insulinindependent tissues such as intestine (Frizzel et al. 1973) and proximal kidney tubule (Ullrich 1976) co-migration of sodium and glucose and/or galactose has been demonstrated. A sugar-concentration gradient would be maintained by endoneurial metabolic
42 p r o c e s s e s t h a t use g l u c o s e a n d / o r g a l a c t o s e (i.e. glycolysis, p o l y o l synthesis via the sorbitol p a t h w a y ,
nonenzymatic
glycosylation, glycogenesis).
Participation
o f the
sorbitol p a t h w a y in the m a i n t e n a n c e o f this s u g a r - c o n c e n t r a t i o n g r a d i e n t c o u l d explain h o w inhibition o f a l d o s e r e d u c t a s e results in d e c r e a s e d e n d o n e u r i a l s o d i u m levels in galactose neuropathy. ACKNOWLEDGEMENTS T h e a u t h o r s gratefully a c k n o w l e d g e t h e e x p e r t t e c h n i c a l a s s i s t a n c e o f M s . H e i d i M. H e c k m a n . T h e a l d o s e r e d u c t a s e inhibitor, I C I 128,436, w a s kindly s u p p l i e d by I C I , Pharmaceutical Division, U.S.A.
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