Effect of Cilostazol on the Neuropathies of Streptozotocin-Induced Diabetic Rats

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Abstract

INTRODUCTION

Both metabolic ^{1, 2)} and vascular defects^3–5) have been implicated in the pathogenesis of diabetic neuropathy, but the precise mechanisms causing peripheral neuropathy have not been elucidated. Nerve conduction is impaired in overt diabetic neuropathy by a combination of structural and metabolic defects in the peripheral nerve⁶⁾. Many studies attribute the slowing of nerve conduction in diabetic rats to alterations in nerve Na⁺ and Na⁺-related metabolism mediated by a reversible Na⁺, K⁺-ATPase defect^{1, 7, 8)}

The decreases in Na⁺, K⁺-ATPase activity and motor nerve conduction velocity (MNCV) in diabetic rats have been reported to be ameliorated by treatment with aldose reductase inhibitors^8–11), dietary myo-inositol supplementation¹²⁾, gangliosides^{13, 14)}, prostaglandin E₁¹⁵⁾, anti-oxidants¹⁶ or essential fatty acids^{17, 18)}. More recent experiments showed that there was a positive correlation between cAMP content and Na⁺,K⁺-ATPase activity, and MNCV and Na⁺,K⁺-ATPase activity in the rat sciatic nerve¹⁹⁾.

Haemodynamic abnormalities of the peripheral nerve have also been suggested as a major cause of the functional deterioration in the neuropathies observed in streptozotocin (STZ)-induced diabetic rats^{3, 20}. In addition to marked biochemical and functional abnormalities, morphological changes have been demonstrated in the nerves of diabetic rats²¹⁾.

Cilostazol(6-[4-cyclohexyl-1H-tetrazol-5-yl)butyl}3,4-dihydro-2(1H)-quinolinone]), a potent phosphodiesterase inhibitor, increases the cAMP content of the sciatic nerves of rats¹⁹⁾. In this study, we examined the effects of cilostazol on biochemical, functional and morphological aspects of experimental diabetic neuropathies induced by STZ in the rat.

MATERIALS and METHODS

RESULTS

Effect of Cilostazol on Body Weight, Blood Glucose and cAMP Contents of Sciatic Nerves

As shown in Table 1, blood glucose levels were significantly elevated and body weights significantly decreased compared to normal control rats 12 weeks after STZ administration. cAMP levels were decreased in the sciatic nerves of the diabetic rats. Administration of cilostazol (weeks 9–12) had no effect on blood glucose levels and body weights but significantly increased cAMP levels of sciatic nerves compared to age-matched, untreated diabetic rats. However, cilostazol treatment did not restore cAMP levels of diabetic rats to those of age-matched normal control rats.

Table 1.

Blood glucose, body weight and cAMP contents of rat sciatic nerves

Effect of Cilostazol on Sciatic Nerve Conduction Velocity (MNCV)

As shown in Table 2, significant reduction in MNCVs were observed in diabetic rats compared to those in age-matched normal rats at the 8th and 12th weeks after STZ treatment (p<0.001). Cilostazol treatment significantly increased MNCV in the sciatic nerves of diabetic rats but they were not restored to the levels noted in age-matched normal control rats.

Table 2.

Motor nerve conduction velocity (MNCV) of rat sciatic nerves at 8 and 12 weeks after streptozotocin treatment

Correlation Between cAMP Contents and MNCV in the Sciatic Nerve

As shown in Fig. 1, there was a positive correlation between cAMP content and MNCV in the sciatic nerve at 12 weeks after STZ treatment (r=0.86; P<0.001).

Fig. 1.

The correlation between cAMP content and MNCV in the sciatic nerve of rats at 12 weeks Eight-week-old, male Sprague-Dawley rats were divided into two groups: an age-matched, normal group and a group rendered diabetic by administration of streptozotocin (STZ; 35mg/kg, i.p.). Eight weeks later, the STZ-treated diabetic rats were further subdivided into two groups: one group consisted of age-matched untreated diabetic rats and, in the second group, the diabetic rats were treated with 0.03% cilostazol in their daily diets for four weeks.

cAMP and MNCV in untreated diabetic rats were scatterd in the lower left part of the curve and cilostazol treatment shifted the data points to the upper right part of the curve.

Morphological Finding

Morphological changes were examined in the sciatic nerve by light and electron microscopy. In the peripheral nerves of the diabetic rat, there was reduction in the number of myelinated fibers and there appeared to be more small fibers in the nerve when compared to non-diabetic control rats. Myelinated fiber density and size distribution were restored after treatment of cilostazol (Fig 2. a, b, c).

Fig. 2.

Light (a, b, c. × 400) and electron (d, e, f. × 5100) photomicrography of semi-thin transverse section of rat sciatic nerves. Eight-week-old, male Sprague-Dawley rats were divided into two groups: an age-matched, normal control (a, d), and a group rendered diabetic by administration of streptozotocin. Eight weeks later, the STZ-treated diabetic rats were further subdivided into two groups: one group was an age-matched untreated diabetic rat (b, e) and the second group was treated with 0.03% cilostazol in their daily diets for four weeks (c, f).

In electron micrographs, a thickening of capillary walls and duplication of basement membranes in the nerves of diabetic rats were noted. These vascular abnormalities were restored towards normal after cilostazol treatment, (Fig 2. c, d, f).

DISCUSSION

The streptozotocin (STZ)-induced diabetic rat is the most commonly used animal model of human diabetic neuropathies. Early changes in nerve function are characterized by reduced nerve conduction velocity in this animal model^{3, 22, 23)}. Diabetic rats of eight weeks’ duration show a significant reduction in sciatic MNCV which can be normalized by insulin treatment and near normoglycemia²⁴⁾. Acute hyperglycemia induced by STZ elicits activation of the polyol pathway with sorbitol accumulation, myo-inositol depletion and reduction of Na⁺,K⁺-ATPase activity^{25, 26)} The decrease in myo-inositol causes depletion of phosphoinositides, followed by poor calcium mobilization and impaired protein kinase C activity, eventually resulting in impaired Na⁺, K⁺-ATPase activity²⁷⁾.

Thus, nerve Na⁺/K⁺ pump activity is reduced; fibers become Na+ loaded, Na⁺ channels become inactivated, and MNCV decreases. Decreasd Na⁺,K⁺ -ATPase activity and motor nerve conduction velocity(MNCV) have been reported to be restored by aldose reductase inhibitors^8–11), dietary myo-inositol supplementation^{8, 12)}, gangliosides,^{13, 14)} prostaglandin E₁¹⁵⁾, anti-oxidants¹⁶⁾ or essential fatty acid^{17, 18)} in diabetic rats. Recent studies have shown that cAMP levels are decreased in the peripheral nerves of STZ-diabetic rats¹⁹⁾. cAMP is one of the intrinsic intracellular modulators which regulates Na⁺,K⁺-ATPse activity or the Na⁺/K⁺ pump in various tissues^{28, 29)}. Therefore, the possibility was suggested that increased cAMP may improve MNCV by restoration of Na⁺,K⁺-ATPse activity. It has been reported that cilostazol increased cAMP content in the nerves of diabetic rats and improved MNCV without changing myo-inositol levels, and the positive correlation between cAMP content and Na+,K⁺-ATPse activity was noted also¹⁹⁾. In addition to marked biochemical and functional abnormalities, morphological changes have been demonstrated in the peripheral nerves of STZ-induced diabetic rats²¹⁾.

As reported by other investigators, STZ in the present study also increased blood glucose levels and reduced body weights of the rats. In addition, the cAMP level and MNCV of sciatic nerves were decreased by STZ as reported earlier.¹⁹ Blood glucose and body weights were unaffected by cilostazol treatment but cAMP levels and MNCV were significantly increased compared to untreated diabetic rats but were not restored to the levels observed in age-matched, normal control animals as previously reported¹⁹⁾. This discrepancy may be explained by the different strains of rats used to produce the diabetic animals or by differences in the duration and/or the severity of the experimental diabetes. Since rats with STZ-induced diabetes were not treated with insulin, we intentionally used rats with a milder diabetic state to ensure survival of the animals to the end of this prolonged study. A positive correlation between cAMP content and MNCV in the sciatic nerve was also observed. Whether the association of a reduced cAMP content with decreased MNCV implies the involvement of cAMP in motor neuron function is unknown at the present time. Reduced cAMP levels could modulate the function of motor neurons and precipitate the neuropathic condition. Clear mechanisms by which cAMP enhances MNCV are unknown, but it is possible that an increased cAMP content could affect cAMP-dependent protein kinase activity, followed by an enhancement of Na⁺,K⁺-ATPase activity in the sciatic nerve. It has been reported that protein kinase C agonists normalized Na⁺,K⁺-ATPase activity in the diabetic rabbit nerve^{27, 30)} and cAMP stimulates protein kinase C activity in cultured renal cells³¹⁾. In addition to various metabolic factors, reduced nerve blood flow, caused by rheological changes coupled with vasa nervorsum microangiopathy, leads to endoneural hypoxia of sufficient magnitude to impair nerve function^{3, 20)}.

Some experimental studies have reported that hyperglycemia-induced blood flow reduction and the resultant endoneural hypoxia were important factors underlying nerve conduction deficits early in the development of diabetic neuropathy³⁾. The alteration of nerve blood flow, accompanied by nerve ischemia in diabetic rats, may be related to functional abnormalities³⁾ and the reduction of Na⁺,K⁺-ATPase activity observed³²⁾. Uehara et al³³⁾ reported that the anti-platelet effects of cilostazol improved nerve blood flow, increased mean myelinated fiber size and enlarged the lumen of endoneural microvessels in diabetic rats. In our study, STZ reduced myelinated fiber size and there appeared to be more small fibers, thickening of capillary walls and duplication of basement membranes in the endoneural microvessels of sciatic nerves when compared to those of the non-diabetic, control rats. The myelinated fiber density, size distribution and vascular abnormalities were restored after treatment of cilostazol. It is possible that correction of nerve ischemia by cilostazol may result in the prevention of structural changes also. Cilostazol is a potent phosphodiesterase inhibitor, which increases the cAMP content in the sciatic nerve of rats¹⁹⁾. Our study revealed that cilostazol can elevate cAMP levels and prevent impairment of MNCV in the sciatic nerves of STZ-induced diabetic rats, functionally and morphologically. This suggests that there is a relationship between the decreased rate of nerve regeneration and the decreased cAMP contents, which may be a possible pathophysiological link in the peripheral motor nerves of diabetic rats. Cilostazol may be useful for the prevention of diabetic neuropathies from functional and structural aspects of its action.

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