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Prevention of abnormalities in motor nerve conduction and nerve blood-flow by a prostacyclin analog, beraprost sodium, in streptozotocin-induced diabetic rats
Prevention of Abnormalities in Motor Nerve Conduction and Nerve Blood-Flow by a Prostacyclin Analog, Beraprost Sodium, in Streptozotocin-Induced Diabetic Rats N. Hotta, N. Koh, F. Sakakibara, J. Nakamura, I’. Hamada, T. Wakao, T. Hara, K. Mori, K. Naruse, K. Nakashima, H. Fukasawa, and H. Kakuta The Third Department of Internal Medicine, of Medicine, Nagoya, Japan
Nagoya University
School
The effects of the prostacyclin analog beraprost sodium on motor nerve function and nerve blood-flow were examined in streptozotocininduced diabetic rats. Oral administration of beraprost sodium 0.1 mg/kglday for 8 wks significantly (p < 0.001) improved caudal motor nerve conduction velocity and sciatic nerve blood-flow, both of which are impaired in diabetic rats. Beraprost sodium did not affect glucose, sorbitol, or fructose levels in the sciatic nerve. However, a decreased content of cyclic AMP in the sciatic nerve and higher level of thromboxane B, in the thoracic aorta of diabetic rats, as compared with those in normal rats, were reversed by the treatment with beraprost sodium (P < 0.01). Results suggest that beraprost sodium may have value in treating diabetic neuropathy, mainly by improving endoneurial blood-flow. Keywords: Prostacyclin; streptozotocin-induced diabetic rats; motor nerve conduction velocity; sciatic nerve blood-flow; thromboxane B,
Introduction Hyperglycemia and the associated metabolic abnormalities in the metabolites of the polyol pathway are thought to contribute to the etiology of Address reprint requests to Nigishi Hotta, M.D., The Third Department of Internal Medicine, Nagoya University School of Medicine, 65 Tsuruma-cho, Showaku, Nagoya 466, Japan.
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diabetic neuropathy. 1-4 There is increasing evidence for a vascular involvement in the pathogenesis of diabetic neuropathy. Reduced peripheral nerve blood-flow and consequent endoneurial hypoxia are major fat tors in the etiology of diabetic neuropathy in both human patients and experimental animals5-15 Recent evidence suggests that microvascular disregulation in diabetes may primarily involve the endothelial-dependent nitric oxide-mediated component, 16t1’contributing to reduce nerve conduction velocity.‘s An impairment of receptor-mediated, endothelial-dependent relaxation of aortic rings from diabetic rabbits and rats has also been reported to be accompanied by an increased generation of endothelium-derived vasoconstrictor prostanoids such as thromboxane A,.19*20 In diabetic rats, a reduction in prostacyclin production in the vasa nervorum seems to depend on a lack of substrate availability,i3 and could be involved in the reduction of nerve blood-flow. A prostacyclin analog, iloprost, prevents the development of abnormalities in motor and sensory nerve conduction velocity in diabetic rats,14 and completely14 or partially15 reverses the existing deficits. However, there is no evidence that prostacyclin treatment may improve the reduced nerve blood-flow and help to prevent nerve dysfunctions. Moreover, the treatment with the prostacyclin analog iloprost in the previous studies was given subcutaneously14 or intraperitoneally15 for diabetic rats, but beraprost sodium can be given orally. The oral administration of the prostacyclin analog beraprost sodium is an advantage from clinical point of view because the long-term administration of this drug is unavoidable for the therapy of chronic complications in diabetic patients. Our objective was to determine whether the oral administration of the prostacyclin analog beraprost sodium21,22 could prevent a reduction in sciatic nerve blood-flow (SNBF) and reduce the elevated vascular level of thromboxane B,, a stable metabolite of thromboxane A,, in rats with streptozotocin-induced diabetes, thereby contributing to an improvement in delayed motor-nerve conduction velocity (MNCV).
Materials and Methods Animals
and Preparations
Seven-week-old male KBL Wistar rats (SPF, Kitayama Lab., Kyoto, Japanj, 190-220 g, were used in the experiments. They received laboratory chow in the form of pellets (CRF-1, Oriental Yeast, Tokyo, Japan), and tap water was freely available. Diabetes was induced by intraperitoneal injection of streptozotocin, 75 mg/kg of body weight. The drug was dissolved in citric acid buffer pH 7.4,l.S mg/mL, immediately before injection.” After receiving streptozotocin for 2 wks diabetic rats were selected at random and divided into two groups. A diabetic control group (group DC,
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n = 10) had free access to laboratory chow and water without treatment for 8 wks. The remaining diabetic rats also had free access to laboratory chow and water, and were administrated beraprost sodium, 0.1 mg/kg/ day, dissolved in distilled water, given orally by gavage daily for 8 wks (group DB, n = 11). One group of normal rats was maintained on beraprost sodium, 0.1 mg/kg/day (group NB, n = 15) in the same manner as the diabetic rats and had free access to laboratory chow and water. Normal controls (group NC, n = 12) received no treatment and had free access to laboratory chow and water for 8 wks. MNCV and SNBF were measured 8 wks after the initiation of treatment. On the next day, blood was sampled from the heart to measure serum glucose and serum lipids, and then we excised the sciatic nerve to measure free sugars and cyclic AMP, and the thoracic aorta to determine thromboxane B, from each diethyl ether-anesthetized rats. Measurements
of MNCV and SNBF
The MNCV was measured in the most rapidly conducting fibers of the tail nerve that supplied the segmental muscle using the method of Miyoshi and Goto, as previously described. 1o*24Rats were kept on a heated pad at a room temperature of 25°C to maintain a constant rectal temperature of 37°C. After the intraperitoneal injection of sodium pentobarbital (30-40 mg/kg), the MNCV was determined with a Neuropak NEM-3102 instrument (Nihon-Koden, Osaka, Japan). SNBF was measured with an analog recorder BW-24 (Biochemical Science, Kanazawa, Japan) from an electrolysis tissue blood-flow meter RBA-2 (Biochemical Science), as described previously.” After incising the femur of each anesthetized rat and exposing the sciatic nerve, we inserted the tip of a needle electrode BENS 200-30 (Biochemical Science), and the hydrogen gas generated by electrolysis at that site was analyzed from disappearance curves during a constant time interval. SNBF was measured at constant room temperature of 25°C in the same room as that used for physiological investigations. Values of SNBF were calculated according to the equation of Koshu et a1.25 Derminations of Blood Glucose, Nerve Free Sugars and Cyclic AMP, and Aortic Thromboxane B, The sciatic nerve was excised from the femur of each diethyl etheranesthetized rat (Katayama Chemical Co., Tokyo, Japan), weighed immediately, and frozen at - 70°C until polyol content and cyclic AMP levels were determined. For measurement of nerve free sugars contents, the sciatic nerve was homogenized in 0.5 mL of cold 8% perchloric acid. After centrifugation at 12000 rpm for 5 min, the supernatants were neutralized with 2N KOH.
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The neutralized extracts were centrifuged at 3000 rpm and the supematants were used for fluorometric assay of free sugars with a MASCO Spectrofluorometer FP-777 (JASCO Co., Tokyo, Japan). Sorbitol was determined enzymatically by the method of Clements et a1.26 Glucose” and fructose2s were assayed by an enzymatic method. For determination of cyclic AMP, the sciatic nerve was boiled for 5 min with 50 mmol/L sodium acetate buffer (pH 4.0), homogenized with a motor-driven glass homogenizer, and then centrifuged at 2000 rpm for 5 min. The supernatant was used for cyclic AMP determination29 with an RIA kit (Yamasashoyu Co., Choshi, Japan). The thoracic aorta was excised from anesthetized rats, cleaned immediately in cold physiological saline solution including 0.1 M indomethatin and 10 mM EDTA, transferred on filter paper to remove water from the tissue, and then stored at - 70°C for assaying thromboxane B,. To measure thromboxane B2, samples were homogenized in 2 mL of 95% ethanol, rinsed by the addition of an equal volume of 95% ethanol, and then centrifuged at 3000 rpm for 15 min. The supernatant was used for thromboxane B, determination by RIA kit (DuPont NEN Research Products, Boston, MA, USA). To measure serum glucose, we sampled blood from the heart. After centrifugation at 3000 rpm for 10 min, aliquots of serum were subjected to biochemical tests. Serum glucose was determined with an autoanalyzer (Enzyme Electrode Analyzer AS 200, Toyo Jyozo, Tokyo, Japan). Determination of Protein Protein content Lowry et aL3’
in tissue
was measured
by the method
described
by
Drugs Streptozotocin (Wako Pure Chemical Co., Tokyo, Japan) was used to induce diabetes. The test drug beraprost sodium was provided by Kaken Pharmaceutical Co., Tokyo, Japan. Other reagents and enzymes used in this study were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Statistics Data are expressed as mean 5 SE. Differences among groups were detected by analysis of variance (ANOVA) and significance at the 0.05 level was assessed by Scheffe’s S test.
Results Body Weight and Serum Glucose Changes in body weight and serum glucose level in all groups of rats are shown in Table 1. Body weight and serum glucose in normal rats were
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similar in the NC and NB groups. Rats with streptozotocin-induced diabetes (group DC) lost a significant amount of weight, but treatment with beraprost sodium (group DB) had no effect on either weight loss or the severity of hyperglycemia. There were no differences in general status between groups NC and NB during the 8-wk experiment, including water intake. However, a significant increase in water intake was observed in all diabetic rats (DC and DB, 70 + 2 ml/day), as compared with normal rats (NC and NB, 42 + 2 ml/day). Caudal Motor Nerve Conduction Velocity and Sciatic Nerve Blood-Flow Administration of beraprost sodium for 8 wks significantly improved the impaired MNCV in diabetic rats, as compared with that in untreated diabetic rats (P < 0.001, Table 2). There was no difference in MNCV between the three groups of the beraprost sodium-treated and untreated normal rats and the beraprost sodium-treated diabetic rats. Blood-flow in the sciatic nerve was the lowest in the DC group (4.5 + 0.5 mL/min/lOO g), which showed a significant decrease in SNBF as compared with groups NC, NB, and DB. There was no difference in SNBF among the beraprost sodium-treated and untreated normal rats and the beraprost sodiumtreated diabetic rats. The data in Table 2 show an inhibitory effect of beraprost sodium on the development of impairments in MNCV and SNBF in diabetic rats. Glucose, Sorbitol, and Fructose Concentrations in the Sciatic Nerve Glucose, sorbitol, and fructose concentrations in the sciatic nerve were markedly elevated in the untreated diabetic rats (group DC) and differed significantly from those in the normal rat groups NC and NB (Table 3). There were no significant differences between groups NC and NB or between groups DC and DB. TABLE
1.
Body weight and serum glucose concentrations
Group/treatment Normal rats (n = 12) Normal rats + beraprost (n = 15) Diabetic rats (n = 10) Diabetic rats + beraprost (n = 11)
Code NC NB DC DB
Body weight (9) 516 515 336 314
f r k k
13 10 9 11
Serum glucose (mmol/L) 5.2 5.3 17.1 16.3
2 + + 2
0.1 0.1 0.6 0.4
Values are mean + SE. Beraprost, beraprost sodium. In all cases, P < 0.001 for differences between values for groups NC and DC, NC and DB, NB and DC, and NB and DB.
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TABLE 2. Caudal motor nerve conduction normal and diabetic rats
velocity
(MNCV) and sciatic nerve blood flow of
8 wks
Group/treatment
MNCV (m/set)
Code
Normal rats (n = 12) Normal rats + beraprost (n = 15) Diabetic rats (n = 10) Diabetic rats + beraprost (n = 11)
NC NB DC DB
‘P < 0.001 versus NC, NB and DB. Values are mean ? SE. Beraprost, beraprost velocity; SNBF, sciatic nerve blood flow.
35.5 35.6 26.1 35.8
sodium;
___ SNBF (ml/min/lOOg)
t 0.6 t- 0.6 -e 0.6’ 2 0.9
MNCV,
motor
12.1 12.3 4.5 13.0
+ I ” 2
0.9 0.7 o.58 0.7
nerve conduction
Concentrations of Cyclic AMP in the Sciatic Nerve and of Thromboxane B, in the Thoracic Aorta As shown in Table 4, the concentration of cyclic AMP in the sciatic nerve of diabetic rats was markedly lower than that of the two groups of normal rats (NC and NB). However, the concentration of cyclic AMP in the sciatic nerve of diabetic rats was restored by treatment with beraprost sodium (group DB, P < 0.01). The concentration of thromboxane B, in the thoracic aorta of diabetic rats was significantly higher than that of the normal rat groups NC and NB. Administration of beraprost sodium significantly reduced the level of thromboxane B, in the thoracic aorta of diabetic rats, as compared with that in untreated diabetic rats (P < 0.05. Table 4).
TABLE 3.
Free sugars in sciatic
nerve
8 wks Group/treatment Normal rats (n = 12) Normal rats + beraprost (n = 15) Diabetic rats (n = 10) Diabetic rats + beraprost (n = 11)
Code NC NB DC DB
‘P < 0.001 versus NC and NB. Values are mean ? SE of sugars concentration, beraprost sodium.
344
Glucose 472 454 789 843
2 2 2 -t
42 39 73’ 45’
nmol/lOO
-.
Sorbitol 33 32 162 141
t- 3 ?I 2 i 6’ t ga
mg of wet weight.
Prostaglandins
Fructose 101 _’8 10729 513 2 338 524 + 29”
Beraprost,
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Thromboxane
Hotta et al.
B2 in thoracic aorta and cyclic AMP in sciatic nerve
8 wks
Group/treatment Normal rats (n = 12) Normal rats + beraprost (n = 15) Diabetic rats (n = 10) Diabetic rats + beraprost (n = 11)
Code NC NB DC DB
TXB, in aorta (pg/mg wet weight) 9.27 8.80 12.78 9.75
-t 1.77 + 1.71 2 1.58e,b -c 1.32
‘P < 0.001 versus NC and NB. P < 0.01 versus DB. Values are mean 2 SE. Beraprost, beraprost sodium, TXB,, thromboxane AMP.
CAMP in nerve (pmollg protein) 1.28 1.23 0.96 1.12
2 + f *
0.05 0.04 0.04erb 0.07
B,; CAMP, cyclic
Discussion The present study showed clearly that treatment with beraprost sodium improved the impaired MNCV and increased the previously decreased sciatic nerve blood-flow in rats with experimental diabetes. These effects occurred in the absence of any effects on nerve polyol pathway metabolites. It is, therefore, unlikely that beraprost sodium treatment modulated the activity of the polyol pathway, which is also related to the development of diabetic neuropathy. Several studies have demonstrated that prostacyclin (prostaglandin Ia) may play a role in the development of diabetic neuropathy. Ward et a1.13 observed a deficit in prostacyclin production by sciatic nerve peri/ epineurial blood vessels in rats with chronic experimental diabetes that presumably contributed to a reduction in nerve blood-flow. The prostacyclin analog iloprost improves vascular function in patients with various types of peripheral vascular disease. 31~32Shindo et al. reported that, in an open, non-randomized clinical trial of iloprost, subjective symptoms such as pain were alleviated in patients with diabetic neuropathy.33 Cotter et al. l4 observed that iloprost treatment largely prevented and reversed the nerve dysfunction in rats with chronic streptozotocin-induced diabetes. These observations suggest strongly that prostacyclin could have therapeutic value in treating diabetic neuropathy. However, the effect of prostacyclin on diabetic neuropathy has not been investigated with respect to nerve function and nerve blood-flow. As shown in Table 2, beraprost sodium significantly improved the impaired MNCV in diabetic rats that was accompanied by a marked increase in previously reduced SNBF. However, there was no increase of SNBF in normal rats by treatment with beraprost sodium while its increase was observed in diabetic rats. The regulatory system of peripheral blood-flow by beraprost sodium may be
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related to at least two effects by the direct relaxation of vascular muscle and the modification of adrenergic nerve activity22 as prostacyclin has a synergic effect with a-blocker. 34 There was a partial disappearance of the sympathetic and parasympathetic nerve endings in the muscle of diabetic patients. 35 Considering these facts, our findings suggest that the autoregulatory system of vessels concerned with vasoconstriction and vasodilation may respond well to the treatment by beraprost sodium in normal rats, but not in diabetic rats, resulting in an increase of SNBF in diabetic rats without its change in normal rats. However, beraprost sodium exerted no effect on nerve polyol metabolites (Table 3), as was the case with iloprost. l4 Thus it is likely that the effect of beraprost sodium on diabetic neuropathy is mediated via vascular factors. Beraprost sodium is known to have a vasodilatory effect that is derived from its interference with the release of Ca2+ from intracellular stores and its transmembrane influx via the receptor-operated process”; it also exerts anti-platelet action in rats.i6 In our study, improvement of these vascular factors by beraprost sodium administration may have contributed to the reversal of the decrease of SNBF in diabetic rats, thus resulting in an improvement of delayed MNCV. Beraprost sodium significantly reduced the elevated level of thromboxane B, in the thoracic aorta of diabetic rats. This finding supports the possibility that an elevated production of vasodilator prostaglandins, accompanied by a decreased level of thromboxane B,, contributes to an increase in previously reduced nerve blood-flow, leading to an improvement in the impaired MNCV in diabetes.36 In our study, beraprost sodium treatment restored to normal sciatic nerve blood-flow in diabetic rats, as assessed by hydrogen polarography. This technique discriminates between nutritive endoneurial and nonnutritive peri/epineurial flow. 11t3’It is clear that beraprost sodium treatment restored sciatic endoneurial blood-flow in diabetic rats. The idea that endoneurial hypoxia is an important determinant of nerve abnormalities is supported by the results of studies in non-diabetic rats chronically exposed to a hypoxic environment, 6r38 in patients with central hypoxemia,3p and in the morphological changes in sural nerves in diabetic and hypoxic patients.40 A rapid improvement in peroneal MNCV was observed after bypass surgery in patients with peripheral vascular disease.4 These findings suggest that treatment with drugs, such as prostacyclin, that could improve blood-flow may also improve nerve function. The reduction of cyclic AMP content in the sciatic nerve of diabetic rats in the present study was prevented by treatment with a prostacyclin analog, beraprost sodium. Reduction of cyclic AMP in the nerves of diabetic rats is reportedly mainly due to impaired adenyl cyclase activity, and may be implicated in the pathogenesis of diabetic neuropathy.42 The
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effect of beraprost sodium on cyclic AMP in the sciatic nerve may offer another possibility for preventing diabetic neuropathy. In conclusion, our results strongly suggest that beraprost sodium or other prostacyclin analog could have therapeutic value in treating diabetic neuropathy, mainly via improvement of the endoneurial bloodflow.
Acknowledgements This research was supported in part by a Diabetes Research grant from the Ministry of Health and Welfare of Japan.
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Editor:
F. Coceani
Prostaglandins 1995:49, June
Received:
l-4-95
Accepted:
5-8-95
349
Nagoya University
School
The effects of the prostacyclin analog beraprost sodium on motor nerve function and nerve blood-flow were examined in streptozotocininduced diabetic rats. Oral administration of beraprost sodium 0.1 mg/kglday for 8 wks significantly (p < 0.001) improved caudal motor nerve conduction velocity and sciatic nerve blood-flow, both of which are impaired in diabetic rats. Beraprost sodium did not affect glucose, sorbitol, or fructose levels in the sciatic nerve. However, a decreased content of cyclic AMP in the sciatic nerve and higher level of thromboxane B, in the thoracic aorta of diabetic rats, as compared with those in normal rats, were reversed by the treatment with beraprost sodium (P < 0.01). Results suggest that beraprost sodium may have value in treating diabetic neuropathy, mainly by improving endoneurial blood-flow. Keywords: Prostacyclin; streptozotocin-induced diabetic rats; motor nerve conduction velocity; sciatic nerve blood-flow; thromboxane B,
Introduction Hyperglycemia and the associated metabolic abnormalities in the metabolites of the polyol pathway are thought to contribute to the etiology of Address reprint requests to Nigishi Hotta, M.D., The Third Department of Internal Medicine, Nagoya University School of Medicine, 65 Tsuruma-cho, Showaku, Nagoya 466, Japan.
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diabetic neuropathy. 1-4 There is increasing evidence for a vascular involvement in the pathogenesis of diabetic neuropathy. Reduced peripheral nerve blood-flow and consequent endoneurial hypoxia are major fat tors in the etiology of diabetic neuropathy in both human patients and experimental animals5-15 Recent evidence suggests that microvascular disregulation in diabetes may primarily involve the endothelial-dependent nitric oxide-mediated component, 16t1’contributing to reduce nerve conduction velocity.‘s An impairment of receptor-mediated, endothelial-dependent relaxation of aortic rings from diabetic rabbits and rats has also been reported to be accompanied by an increased generation of endothelium-derived vasoconstrictor prostanoids such as thromboxane A,.19*20 In diabetic rats, a reduction in prostacyclin production in the vasa nervorum seems to depend on a lack of substrate availability,i3 and could be involved in the reduction of nerve blood-flow. A prostacyclin analog, iloprost, prevents the development of abnormalities in motor and sensory nerve conduction velocity in diabetic rats,14 and completely14 or partially15 reverses the existing deficits. However, there is no evidence that prostacyclin treatment may improve the reduced nerve blood-flow and help to prevent nerve dysfunctions. Moreover, the treatment with the prostacyclin analog iloprost in the previous studies was given subcutaneously14 or intraperitoneally15 for diabetic rats, but beraprost sodium can be given orally. The oral administration of the prostacyclin analog beraprost sodium is an advantage from clinical point of view because the long-term administration of this drug is unavoidable for the therapy of chronic complications in diabetic patients. Our objective was to determine whether the oral administration of the prostacyclin analog beraprost sodium21,22 could prevent a reduction in sciatic nerve blood-flow (SNBF) and reduce the elevated vascular level of thromboxane B,, a stable metabolite of thromboxane A,, in rats with streptozotocin-induced diabetes, thereby contributing to an improvement in delayed motor-nerve conduction velocity (MNCV).
Materials and Methods Animals
and Preparations
Seven-week-old male KBL Wistar rats (SPF, Kitayama Lab., Kyoto, Japanj, 190-220 g, were used in the experiments. They received laboratory chow in the form of pellets (CRF-1, Oriental Yeast, Tokyo, Japan), and tap water was freely available. Diabetes was induced by intraperitoneal injection of streptozotocin, 75 mg/kg of body weight. The drug was dissolved in citric acid buffer pH 7.4,l.S mg/mL, immediately before injection.” After receiving streptozotocin for 2 wks diabetic rats were selected at random and divided into two groups. A diabetic control group (group DC,
340
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Hotta et al.
n = 10) had free access to laboratory chow and water without treatment for 8 wks. The remaining diabetic rats also had free access to laboratory chow and water, and were administrated beraprost sodium, 0.1 mg/kg/ day, dissolved in distilled water, given orally by gavage daily for 8 wks (group DB, n = 11). One group of normal rats was maintained on beraprost sodium, 0.1 mg/kg/day (group NB, n = 15) in the same manner as the diabetic rats and had free access to laboratory chow and water. Normal controls (group NC, n = 12) received no treatment and had free access to laboratory chow and water for 8 wks. MNCV and SNBF were measured 8 wks after the initiation of treatment. On the next day, blood was sampled from the heart to measure serum glucose and serum lipids, and then we excised the sciatic nerve to measure free sugars and cyclic AMP, and the thoracic aorta to determine thromboxane B, from each diethyl ether-anesthetized rats. Measurements
of MNCV and SNBF
The MNCV was measured in the most rapidly conducting fibers of the tail nerve that supplied the segmental muscle using the method of Miyoshi and Goto, as previously described. 1o*24Rats were kept on a heated pad at a room temperature of 25°C to maintain a constant rectal temperature of 37°C. After the intraperitoneal injection of sodium pentobarbital (30-40 mg/kg), the MNCV was determined with a Neuropak NEM-3102 instrument (Nihon-Koden, Osaka, Japan). SNBF was measured with an analog recorder BW-24 (Biochemical Science, Kanazawa, Japan) from an electrolysis tissue blood-flow meter RBA-2 (Biochemical Science), as described previously.” After incising the femur of each anesthetized rat and exposing the sciatic nerve, we inserted the tip of a needle electrode BENS 200-30 (Biochemical Science), and the hydrogen gas generated by electrolysis at that site was analyzed from disappearance curves during a constant time interval. SNBF was measured at constant room temperature of 25°C in the same room as that used for physiological investigations. Values of SNBF were calculated according to the equation of Koshu et a1.25 Derminations of Blood Glucose, Nerve Free Sugars and Cyclic AMP, and Aortic Thromboxane B, The sciatic nerve was excised from the femur of each diethyl etheranesthetized rat (Katayama Chemical Co., Tokyo, Japan), weighed immediately, and frozen at - 70°C until polyol content and cyclic AMP levels were determined. For measurement of nerve free sugars contents, the sciatic nerve was homogenized in 0.5 mL of cold 8% perchloric acid. After centrifugation at 12000 rpm for 5 min, the supernatants were neutralized with 2N KOH.
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Beraprost
sodium
and diabetic
rats:
Hotta et al.
The neutralized extracts were centrifuged at 3000 rpm and the supematants were used for fluorometric assay of free sugars with a MASCO Spectrofluorometer FP-777 (JASCO Co., Tokyo, Japan). Sorbitol was determined enzymatically by the method of Clements et a1.26 Glucose” and fructose2s were assayed by an enzymatic method. For determination of cyclic AMP, the sciatic nerve was boiled for 5 min with 50 mmol/L sodium acetate buffer (pH 4.0), homogenized with a motor-driven glass homogenizer, and then centrifuged at 2000 rpm for 5 min. The supernatant was used for cyclic AMP determination29 with an RIA kit (Yamasashoyu Co., Choshi, Japan). The thoracic aorta was excised from anesthetized rats, cleaned immediately in cold physiological saline solution including 0.1 M indomethatin and 10 mM EDTA, transferred on filter paper to remove water from the tissue, and then stored at - 70°C for assaying thromboxane B,. To measure thromboxane B2, samples were homogenized in 2 mL of 95% ethanol, rinsed by the addition of an equal volume of 95% ethanol, and then centrifuged at 3000 rpm for 15 min. The supernatant was used for thromboxane B, determination by RIA kit (DuPont NEN Research Products, Boston, MA, USA). To measure serum glucose, we sampled blood from the heart. After centrifugation at 3000 rpm for 10 min, aliquots of serum were subjected to biochemical tests. Serum glucose was determined with an autoanalyzer (Enzyme Electrode Analyzer AS 200, Toyo Jyozo, Tokyo, Japan). Determination of Protein Protein content Lowry et aL3’
in tissue
was measured
by the method
described
by
Drugs Streptozotocin (Wako Pure Chemical Co., Tokyo, Japan) was used to induce diabetes. The test drug beraprost sodium was provided by Kaken Pharmaceutical Co., Tokyo, Japan. Other reagents and enzymes used in this study were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Statistics Data are expressed as mean 5 SE. Differences among groups were detected by analysis of variance (ANOVA) and significance at the 0.05 level was assessed by Scheffe’s S test.
Results Body Weight and Serum Glucose Changes in body weight and serum glucose level in all groups of rats are shown in Table 1. Body weight and serum glucose in normal rats were
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similar in the NC and NB groups. Rats with streptozotocin-induced diabetes (group DC) lost a significant amount of weight, but treatment with beraprost sodium (group DB) had no effect on either weight loss or the severity of hyperglycemia. There were no differences in general status between groups NC and NB during the 8-wk experiment, including water intake. However, a significant increase in water intake was observed in all diabetic rats (DC and DB, 70 + 2 ml/day), as compared with normal rats (NC and NB, 42 + 2 ml/day). Caudal Motor Nerve Conduction Velocity and Sciatic Nerve Blood-Flow Administration of beraprost sodium for 8 wks significantly improved the impaired MNCV in diabetic rats, as compared with that in untreated diabetic rats (P < 0.001, Table 2). There was no difference in MNCV between the three groups of the beraprost sodium-treated and untreated normal rats and the beraprost sodium-treated diabetic rats. Blood-flow in the sciatic nerve was the lowest in the DC group (4.5 + 0.5 mL/min/lOO g), which showed a significant decrease in SNBF as compared with groups NC, NB, and DB. There was no difference in SNBF among the beraprost sodium-treated and untreated normal rats and the beraprost sodiumtreated diabetic rats. The data in Table 2 show an inhibitory effect of beraprost sodium on the development of impairments in MNCV and SNBF in diabetic rats. Glucose, Sorbitol, and Fructose Concentrations in the Sciatic Nerve Glucose, sorbitol, and fructose concentrations in the sciatic nerve were markedly elevated in the untreated diabetic rats (group DC) and differed significantly from those in the normal rat groups NC and NB (Table 3). There were no significant differences between groups NC and NB or between groups DC and DB. TABLE
1.
Body weight and serum glucose concentrations
Group/treatment Normal rats (n = 12) Normal rats + beraprost (n = 15) Diabetic rats (n = 10) Diabetic rats + beraprost (n = 11)
Code NC NB DC DB
Body weight (9) 516 515 336 314
f r k k
13 10 9 11
Serum glucose (mmol/L) 5.2 5.3 17.1 16.3
2 + + 2
0.1 0.1 0.6 0.4
Values are mean + SE. Beraprost, beraprost sodium. In all cases, P < 0.001 for differences between values for groups NC and DC, NC and DB, NB and DC, and NB and DB.
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TABLE 2. Caudal motor nerve conduction normal and diabetic rats
velocity
(MNCV) and sciatic nerve blood flow of
8 wks
Group/treatment
MNCV (m/set)
Code
Normal rats (n = 12) Normal rats + beraprost (n = 15) Diabetic rats (n = 10) Diabetic rats + beraprost (n = 11)
NC NB DC DB
‘P < 0.001 versus NC, NB and DB. Values are mean ? SE. Beraprost, beraprost velocity; SNBF, sciatic nerve blood flow.
35.5 35.6 26.1 35.8
sodium;
___ SNBF (ml/min/lOOg)
t 0.6 t- 0.6 -e 0.6’ 2 0.9
MNCV,
motor
12.1 12.3 4.5 13.0
+ I ” 2
0.9 0.7 o.58 0.7
nerve conduction
Concentrations of Cyclic AMP in the Sciatic Nerve and of Thromboxane B, in the Thoracic Aorta As shown in Table 4, the concentration of cyclic AMP in the sciatic nerve of diabetic rats was markedly lower than that of the two groups of normal rats (NC and NB). However, the concentration of cyclic AMP in the sciatic nerve of diabetic rats was restored by treatment with beraprost sodium (group DB, P < 0.01). The concentration of thromboxane B, in the thoracic aorta of diabetic rats was significantly higher than that of the normal rat groups NC and NB. Administration of beraprost sodium significantly reduced the level of thromboxane B, in the thoracic aorta of diabetic rats, as compared with that in untreated diabetic rats (P < 0.05. Table 4).
TABLE 3.
Free sugars in sciatic
nerve
8 wks Group/treatment Normal rats (n = 12) Normal rats + beraprost (n = 15) Diabetic rats (n = 10) Diabetic rats + beraprost (n = 11)
Code NC NB DC DB
‘P < 0.001 versus NC and NB. Values are mean ? SE of sugars concentration, beraprost sodium.
344
Glucose 472 454 789 843
2 2 2 -t
42 39 73’ 45’
nmol/lOO
-.
Sorbitol 33 32 162 141
t- 3 ?I 2 i 6’ t ga
mg of wet weight.
Prostaglandins
Fructose 101 _’8 10729 513 2 338 524 + 29”
Beraprost,
1995:49,
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Beraprost sodium and diabetic ra?: TABLE 4.
Thromboxane
Hotta et al.
B2 in thoracic aorta and cyclic AMP in sciatic nerve
8 wks
Group/treatment Normal rats (n = 12) Normal rats + beraprost (n = 15) Diabetic rats (n = 10) Diabetic rats + beraprost (n = 11)
Code NC NB DC DB
TXB, in aorta (pg/mg wet weight) 9.27 8.80 12.78 9.75
-t 1.77 + 1.71 2 1.58e,b -c 1.32
‘P < 0.001 versus NC and NB. P < 0.01 versus DB. Values are mean 2 SE. Beraprost, beraprost sodium, TXB,, thromboxane AMP.
CAMP in nerve (pmollg protein) 1.28 1.23 0.96 1.12
2 + f *
0.05 0.04 0.04erb 0.07
B,; CAMP, cyclic
Discussion The present study showed clearly that treatment with beraprost sodium improved the impaired MNCV and increased the previously decreased sciatic nerve blood-flow in rats with experimental diabetes. These effects occurred in the absence of any effects on nerve polyol pathway metabolites. It is, therefore, unlikely that beraprost sodium treatment modulated the activity of the polyol pathway, which is also related to the development of diabetic neuropathy. Several studies have demonstrated that prostacyclin (prostaglandin Ia) may play a role in the development of diabetic neuropathy. Ward et a1.13 observed a deficit in prostacyclin production by sciatic nerve peri/ epineurial blood vessels in rats with chronic experimental diabetes that presumably contributed to a reduction in nerve blood-flow. The prostacyclin analog iloprost improves vascular function in patients with various types of peripheral vascular disease. 31~32Shindo et al. reported that, in an open, non-randomized clinical trial of iloprost, subjective symptoms such as pain were alleviated in patients with diabetic neuropathy.33 Cotter et al. l4 observed that iloprost treatment largely prevented and reversed the nerve dysfunction in rats with chronic streptozotocin-induced diabetes. These observations suggest strongly that prostacyclin could have therapeutic value in treating diabetic neuropathy. However, the effect of prostacyclin on diabetic neuropathy has not been investigated with respect to nerve function and nerve blood-flow. As shown in Table 2, beraprost sodium significantly improved the impaired MNCV in diabetic rats that was accompanied by a marked increase in previously reduced SNBF. However, there was no increase of SNBF in normal rats by treatment with beraprost sodium while its increase was observed in diabetic rats. The regulatory system of peripheral blood-flow by beraprost sodium may be
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Beraprost sodium and diabetic rats:
Hotta et al.
related to at least two effects by the direct relaxation of vascular muscle and the modification of adrenergic nerve activity22 as prostacyclin has a synergic effect with a-blocker. 34 There was a partial disappearance of the sympathetic and parasympathetic nerve endings in the muscle of diabetic patients. 35 Considering these facts, our findings suggest that the autoregulatory system of vessels concerned with vasoconstriction and vasodilation may respond well to the treatment by beraprost sodium in normal rats, but not in diabetic rats, resulting in an increase of SNBF in diabetic rats without its change in normal rats. However, beraprost sodium exerted no effect on nerve polyol metabolites (Table 3), as was the case with iloprost. l4 Thus it is likely that the effect of beraprost sodium on diabetic neuropathy is mediated via vascular factors. Beraprost sodium is known to have a vasodilatory effect that is derived from its interference with the release of Ca2+ from intracellular stores and its transmembrane influx via the receptor-operated process”; it also exerts anti-platelet action in rats.i6 In our study, improvement of these vascular factors by beraprost sodium administration may have contributed to the reversal of the decrease of SNBF in diabetic rats, thus resulting in an improvement of delayed MNCV. Beraprost sodium significantly reduced the elevated level of thromboxane B, in the thoracic aorta of diabetic rats. This finding supports the possibility that an elevated production of vasodilator prostaglandins, accompanied by a decreased level of thromboxane B,, contributes to an increase in previously reduced nerve blood-flow, leading to an improvement in the impaired MNCV in diabetes.36 In our study, beraprost sodium treatment restored to normal sciatic nerve blood-flow in diabetic rats, as assessed by hydrogen polarography. This technique discriminates between nutritive endoneurial and nonnutritive peri/epineurial flow. 11t3’It is clear that beraprost sodium treatment restored sciatic endoneurial blood-flow in diabetic rats. The idea that endoneurial hypoxia is an important determinant of nerve abnormalities is supported by the results of studies in non-diabetic rats chronically exposed to a hypoxic environment, 6r38 in patients with central hypoxemia,3p and in the morphological changes in sural nerves in diabetic and hypoxic patients.40 A rapid improvement in peroneal MNCV was observed after bypass surgery in patients with peripheral vascular disease.4 These findings suggest that treatment with drugs, such as prostacyclin, that could improve blood-flow may also improve nerve function. The reduction of cyclic AMP content in the sciatic nerve of diabetic rats in the present study was prevented by treatment with a prostacyclin analog, beraprost sodium. Reduction of cyclic AMP in the nerves of diabetic rats is reportedly mainly due to impaired adenyl cyclase activity, and may be implicated in the pathogenesis of diabetic neuropathy.42 The
346
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effect of beraprost sodium on cyclic AMP in the sciatic nerve may offer another possibility for preventing diabetic neuropathy. In conclusion, our results strongly suggest that beraprost sodium or other prostacyclin analog could have therapeutic value in treating diabetic neuropathy, mainly via improvement of the endoneurial bloodflow.
Acknowledgements This research was supported in part by a Diabetes Research grant from the Ministry of Health and Welfare of Japan.
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Editor:
F. Coceani
Prostaglandins 1995:49, June
Received:
l-4-95
Accepted:
5-8-95
349