EXPERIMENTAL
NEWROLOGY
ll!i,
%%-2%
(1992)
Effect of ~~Met~y~cate~~o~ on Sciatic Nerve crofts Factor Level and Motor Nerve Conduction Velocity in Experimental Diabetic Neuropat~ic Process in Rats YASUO HANAOKA,*
TAKEKAZU OHI,* SNOB F~UKAWA,~ YOSHIK~ FWRU~WA,~ KYOZOHAYASWI,$ AND SHIGERU MATSUKURA* *Division of Neurology, Department of internal Medicine, Miyazaki Medieat College, Miyazaki, Japan; f Diuision of ~~urairnrnu~~~, ~~~tit~t~ of ~eurosc~~e, ~ati~~~ Center of ~eu~~~ and Psyck~t~, Kadaira, Japan; and $Department of ~kar~~e~t~s, Gifu P~r~~tical ~~i~ersi~, Cifu, Japan tor neurons might be involved NGF-sensitive neurons during early periods of ontogenesis (10,ll). For example, the sciatic nerve, which contains axons of motor, sympathetic, and sensory neurons, is known to be one of the pathways of NGF transportation (12). Injury to the rat sciatic nerve leads in viuo to dramatic increases in local NGF synthesis and NGF-mRNA expression (12, 13) as well as to accumulation of NGF at the site distal to the injury (14). Since a decreased rate of sciatic nerve regeneration has been reported in STZ-induced diabetic rats (15, 161, this decrease in endogenous NGF levels could be of relevance to the pathogenesis of diabetic neuropathy. Previous studies showed that 4-methylcatechol (4MC), a derivative of ~atecholamine that probably lacks adrenergic action (17,18), stimulates the synthesis and secretion of NGF in cultured cells (17) and sciatic nerve segments (18). We have recently indicated that intraperitoneal administration of 4-MC can induce NGF synthesis in the peripheral organs of sympathetic neurons and that NGF is retrogradely transposed to the neuronal cell bodies in a physiological manner (19, 20). In this present study we investigated the effects of 4-MC on the endogenous NGF level and MNCV in experimental diabetic rats.
This study examined the effects of 4-methylcatechol (4-MC), a nonamine cateehol compound, on the neuropatbic process of streptozotoein {ST’&)-induced diabetic rats. 4-MC is one of the potent stimulators of nerve growth factor (NGF) synthesis at the cellular level and in cultured sciatic nerve segments of rats. Diabetic rats showed a statisticaIly significant fall in sciatic motor nerve conduction velocity (MNCV) and a si~i~c&ntly reduced NGF content in the sciatic nerve (38.5 z!z2.8% of control, P c 0.01) during the experimental period of 4 weeks, 4-M% treatment of the diabetic rats for 4 weeks starting from the STZ injection elevated the NGF content (140% of untreated diabetic rats, P < 0.05) and prevented the reduction in MNCV, but no effect on high blood glucose levels was seen. These findings suggest that decreased NGF levels in the sciatic nerve of the experimental diabetic rat may be involved in the development of the diabetic neuropathie process and that 4-MC, which can elevate endogenous NGF levels in uiuo, may compensate for the inhibitory effect of ST2 on the NGF level in progressive diabetic neuropathy. 0 1992 Academic Press, Inc.
INTRODIJCTION
Diabetic neuropathy is characterized by symptoms and signs of sensory, motor, and autonomic nerve dysfunction (1, 2). The streptozotocin ~STZ~-induced diabetic rat is the most commonly used animal model of human diabetic neuropathy with reduction of motor nerve conduction velocity (MN~V} (3-5). In previous studies, it was demonstrated that a reduction in retrograde axonal transport of nerve growth factor (NGF) (6) and a decrease in end~genous NGF levels (7) occurred in the sciatic nerves of these experimental diabetic rats. NGF is an essential protein for growth and maintenance of peripheral sympathetic and sensory neurons (8,Q). It was also reported that peripheral mo~14-4666/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
MATERIALS Animals
and Indigen
AND METHODS of Diabetes
We used 6-week-old male Spra~e-Dawley rats weighing less than 150 g. Twenty rats were made diabetic by one intraperitoneal injection of STZ in 0.01 M citrate (pH 4.5) at a dosage of 100 mglkg body wt. Seven control rats received an intraperitoneal injection of citrate buffer alone. Rats were accepted as diabetic when the nonfasting plasma glucose concentration exceeded 400 mgldl, which occurred at 3 days and 4 weeks after 292
EFFECT
OF
CMETHYLCATECHOL
the injection. Of the 20 diabetic rats, 9 of them were left untreated and 11 were administered 4-MC. Experimental
Organization
and Treatment
of Animals
4-MC (obtained from Tokyo Kasei Co., Ltd.) was injected intraperitonealy into 11 rats at a dose of 10 fig/kg body wt in phosphate-buffered saline (PBS) daily for 4 weeks, beginning immediately after the STZ injection. The 7 control rats and 9 diabetic rats received a daily intraperitoneal injection of PBS alone. All animals were maintained in conventional cages with free access to rat chow and water. At Weeks 0,2, and 4 of the experiment, the MNCV in the right sciatic nerve was measured. Immediately after the final measurements, 6 control rats, 8 untreated diabetic rats, and all 11 4-MC-administered diabetic rats were killed; and their sciatic nerves were removed and kept at -3O’C until the nerve NGF level could be measured by enzyme immunoassay (EIA). Eleetrophysiolo~~eal
DIABETIC
NEUROPATHY
of NGF Levels (Two-Site
293
ery obtained by the freezing and thawing method exceeded 90%, judged by a comparison with the results of homogenization method. Therefore, values of NGF levels were used without correction. Statistical Analysis
All measurements were carried out in double-blind fashion, and codes were broken only after analysis of the data had been performed. All data were expressed as the mean it SD. Comparison of the data was performed by one-way analysis of variance. P values of MNCVs and NGF levels were less than 0.001 and 0.0001, respectively. 4-MC-administered and untreated diabetic groups were compared with the nonparametric Wilcoxon test. Comparisons of the control group and untreated or 4-Mu-administered diabetic group were performed by the nonparametric Wilcoxon test, respectively. Significant differences were defined as P x 0.05. RESULTS
methods
The method for measurement of MNCV is described in detail elsewhere (4, 23). Rats were anesthetized with pentobarbital sodium (40 mglkgf and the experiments performed in a warm room. The rectal temperature was maintained from 35 to 37’C with a heating lamp and did not differ between the diabetic and control rats. Recordings were made on the right hind limb, which was held in full extension by strapping. The sciatic and tibia1 nerves were stimulated at the sciatic notch and ankle, respectively, by a 0.2-ms square pulse delivered through a pair of monopolar needle electrodes previously inserted through the skin to lie alongside the sciatic nerve. The muscle action potential was recorded from the interosseous muscle of the right hind limb by monopolar needle electrodes. Distal and proximal latencies were measured from photographs of the oscilloscope recordings. Conduction velocity was calculated from an estimate of length of the nerve between the two stimulating points measured on the skin. Determination
ON
EIA)
NGF was determined as previously described in detail (17, 18, 21). Sciatic nerve segments, each of 2-mm length, were taken from right sciatic nerves and rapidly frozen at -30°C. NGF was effectively extracted by repetitive freezing and thawing. The number of freezing/ thawing cycles was optimized as 6, which gave a result comparable with that obtained by a conventional homogenization procedure (18,19). Twenty microliters of tissue extract solution of each segment was directly applied to a sensitive two-site EIA system for mouse fiNGF. The EIA system could detect as low as 1 pg/ml of NGF. NGF content was expressed as picograms of NGF per milligram of wet weight of sciatic nerve. NGF recov-
Clinical Observations
All diabetic rats showed marked impairment of growth. At the beginning of the experiment, the rats weighed 124 -t 11 g. Four weeks later, rats that had received citrate buffer alone weighed 348 rt: 27 g, whereas those given ST2 weighed only 165 f 51 g. Rats given STZ in combination with 4-MC weighed 186 + 53 g, indicating that 4-MC treatment did not have any effect on the body weight of diabetic rats. All diabetic rats showed marked h~erglycemia. 4MC treatment did not have any effect on glucose level (144 + 54 mg/dl in control rats, 607 -t 81 mg/dl in diabetic rats, and 650 +- 135 mg/dl in 4-MC-administered diabetic rats). Electrophysiological Examination
MNCVs at Weeks 0 and 2 were not si~i~cantly different among the three groups. At the end of Week 4, untreated diabetic rats showed no age-related increment of MNCV. The difference in MNCV between control rats (42.1 + 2.4 m/s) and untreated diabetic rats (34.9 + 3.6 m/s) after 4 weeks reached significance (P < 0.05, Fig. 1). 4-MC-administered rats showed an improved MNCV (40.8 & 3.2 m/s) and there was no significant difference between it and that of control rats. MNCVs of 4-MC-administered diabetic rats were significantly faster than those of untreated diabetic rats (P < 0.01). NGF Levels in the Sciatic Nerves
NGF levels at 4 weeks were significantly decreased to 0.15 1- 0.05 and 0.21 + 0.07 pg NGF/mg wet wt in the sciatic nerves of diabetic and 4-MC-administered dia-
294
HANAOKA
11 *** ii
I
0
2 Time
4
ET
AL.
reduction in myelin thickness. A recent study showed that myelin is more severely affected than the axon and that myelin thickness is reduced earlier than axon diameter in ST&induced diabetic rats (5). Diabetic rats show a variety of detrimental alterations in peripheral nerve such as accumulation of sorbitol pathway metabolites (23,24), depletion of myoinositol (25), reduced nerve blood flow, and hypoxia in the endoneurium (26). It is known that slow axonal transport of neurofilaments in peripheral nerves is reduced in STZ-induced diabetic rats (27). Accumulation of choline acetyltransferase activity proximal to a sciatic nerve crush was reduced in experimental diabetes in rats (28). Furthermore, several groups have found that diabetes causes alterations in fast and slow orthograde and retrograde transport in several classes of neurons (29-31). It was demonstrated that the retrograde axonal transport of ‘251-labeled NGF was reduced in experimental diabetic rats (6). A recent study has shown that endogenous levels of NGF in sciatic nerves of STZ-in-
(weeks)
FIG. 1. Effect of 4-MC on MNCV in the sciatic nerve. MNCVs of 4-MC-admi~stered diabetic rats (e, n = 11) were significantly faster than those of untreated diabetic rats (A, n = 9) (P .c O.Ol), but no significant difference was found between them and those of control rats (0, n = 7) at 4 weeks from the start of STZ injection. MNCVs of untreated diabetic rats were significantly slower than those of control rats (P < 0.05). The values represent the mean f SD. *P < 0.01; **P
NEWROLOGY
ll!i,
%%-2%
(1992)
Effect of ~~Met~y~cate~~o~ on Sciatic Nerve crofts Factor Level and Motor Nerve Conduction Velocity in Experimental Diabetic Neuropat~ic Process in Rats YASUO HANAOKA,*
TAKEKAZU OHI,* SNOB F~UKAWA,~ YOSHIK~ FWRU~WA,~ KYOZOHAYASWI,$ AND SHIGERU MATSUKURA* *Division of Neurology, Department of internal Medicine, Miyazaki Medieat College, Miyazaki, Japan; f Diuision of ~~urairnrnu~~~, ~~~tit~t~ of ~eurosc~~e, ~ati~~~ Center of ~eu~~~ and Psyck~t~, Kadaira, Japan; and $Department of ~kar~~e~t~s, Gifu P~r~~tical ~~i~ersi~, Cifu, Japan tor neurons might be involved NGF-sensitive neurons during early periods of ontogenesis (10,ll). For example, the sciatic nerve, which contains axons of motor, sympathetic, and sensory neurons, is known to be one of the pathways of NGF transportation (12). Injury to the rat sciatic nerve leads in viuo to dramatic increases in local NGF synthesis and NGF-mRNA expression (12, 13) as well as to accumulation of NGF at the site distal to the injury (14). Since a decreased rate of sciatic nerve regeneration has been reported in STZ-induced diabetic rats (15, 161, this decrease in endogenous NGF levels could be of relevance to the pathogenesis of diabetic neuropathy. Previous studies showed that 4-methylcatechol (4MC), a derivative of ~atecholamine that probably lacks adrenergic action (17,18), stimulates the synthesis and secretion of NGF in cultured cells (17) and sciatic nerve segments (18). We have recently indicated that intraperitoneal administration of 4-MC can induce NGF synthesis in the peripheral organs of sympathetic neurons and that NGF is retrogradely transposed to the neuronal cell bodies in a physiological manner (19, 20). In this present study we investigated the effects of 4-MC on the endogenous NGF level and MNCV in experimental diabetic rats.
This study examined the effects of 4-methylcatechol (4-MC), a nonamine cateehol compound, on the neuropatbic process of streptozotoein {ST’&)-induced diabetic rats. 4-MC is one of the potent stimulators of nerve growth factor (NGF) synthesis at the cellular level and in cultured sciatic nerve segments of rats. Diabetic rats showed a statisticaIly significant fall in sciatic motor nerve conduction velocity (MNCV) and a si~i~c&ntly reduced NGF content in the sciatic nerve (38.5 z!z2.8% of control, P c 0.01) during the experimental period of 4 weeks, 4-M% treatment of the diabetic rats for 4 weeks starting from the STZ injection elevated the NGF content (140% of untreated diabetic rats, P < 0.05) and prevented the reduction in MNCV, but no effect on high blood glucose levels was seen. These findings suggest that decreased NGF levels in the sciatic nerve of the experimental diabetic rat may be involved in the development of the diabetic neuropathie process and that 4-MC, which can elevate endogenous NGF levels in uiuo, may compensate for the inhibitory effect of ST2 on the NGF level in progressive diabetic neuropathy. 0 1992 Academic Press, Inc.
INTRODIJCTION
Diabetic neuropathy is characterized by symptoms and signs of sensory, motor, and autonomic nerve dysfunction (1, 2). The streptozotocin ~STZ~-induced diabetic rat is the most commonly used animal model of human diabetic neuropathy with reduction of motor nerve conduction velocity (MN~V} (3-5). In previous studies, it was demonstrated that a reduction in retrograde axonal transport of nerve growth factor (NGF) (6) and a decrease in end~genous NGF levels (7) occurred in the sciatic nerves of these experimental diabetic rats. NGF is an essential protein for growth and maintenance of peripheral sympathetic and sensory neurons (8,Q). It was also reported that peripheral mo~14-4666/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
MATERIALS Animals
and Indigen
AND METHODS of Diabetes
We used 6-week-old male Spra~e-Dawley rats weighing less than 150 g. Twenty rats were made diabetic by one intraperitoneal injection of STZ in 0.01 M citrate (pH 4.5) at a dosage of 100 mglkg body wt. Seven control rats received an intraperitoneal injection of citrate buffer alone. Rats were accepted as diabetic when the nonfasting plasma glucose concentration exceeded 400 mgldl, which occurred at 3 days and 4 weeks after 292
EFFECT
OF
CMETHYLCATECHOL
the injection. Of the 20 diabetic rats, 9 of them were left untreated and 11 were administered 4-MC. Experimental
Organization
and Treatment
of Animals
4-MC (obtained from Tokyo Kasei Co., Ltd.) was injected intraperitonealy into 11 rats at a dose of 10 fig/kg body wt in phosphate-buffered saline (PBS) daily for 4 weeks, beginning immediately after the STZ injection. The 7 control rats and 9 diabetic rats received a daily intraperitoneal injection of PBS alone. All animals were maintained in conventional cages with free access to rat chow and water. At Weeks 0,2, and 4 of the experiment, the MNCV in the right sciatic nerve was measured. Immediately after the final measurements, 6 control rats, 8 untreated diabetic rats, and all 11 4-MC-administered diabetic rats were killed; and their sciatic nerves were removed and kept at -3O’C until the nerve NGF level could be measured by enzyme immunoassay (EIA). Eleetrophysiolo~~eal
DIABETIC
NEUROPATHY
of NGF Levels (Two-Site
293
ery obtained by the freezing and thawing method exceeded 90%, judged by a comparison with the results of homogenization method. Therefore, values of NGF levels were used without correction. Statistical Analysis
All measurements were carried out in double-blind fashion, and codes were broken only after analysis of the data had been performed. All data were expressed as the mean it SD. Comparison of the data was performed by one-way analysis of variance. P values of MNCVs and NGF levels were less than 0.001 and 0.0001, respectively. 4-MC-administered and untreated diabetic groups were compared with the nonparametric Wilcoxon test. Comparisons of the control group and untreated or 4-Mu-administered diabetic group were performed by the nonparametric Wilcoxon test, respectively. Significant differences were defined as P x 0.05. RESULTS
methods
The method for measurement of MNCV is described in detail elsewhere (4, 23). Rats were anesthetized with pentobarbital sodium (40 mglkgf and the experiments performed in a warm room. The rectal temperature was maintained from 35 to 37’C with a heating lamp and did not differ between the diabetic and control rats. Recordings were made on the right hind limb, which was held in full extension by strapping. The sciatic and tibia1 nerves were stimulated at the sciatic notch and ankle, respectively, by a 0.2-ms square pulse delivered through a pair of monopolar needle electrodes previously inserted through the skin to lie alongside the sciatic nerve. The muscle action potential was recorded from the interosseous muscle of the right hind limb by monopolar needle electrodes. Distal and proximal latencies were measured from photographs of the oscilloscope recordings. Conduction velocity was calculated from an estimate of length of the nerve between the two stimulating points measured on the skin. Determination
ON
EIA)
NGF was determined as previously described in detail (17, 18, 21). Sciatic nerve segments, each of 2-mm length, were taken from right sciatic nerves and rapidly frozen at -30°C. NGF was effectively extracted by repetitive freezing and thawing. The number of freezing/ thawing cycles was optimized as 6, which gave a result comparable with that obtained by a conventional homogenization procedure (18,19). Twenty microliters of tissue extract solution of each segment was directly applied to a sensitive two-site EIA system for mouse fiNGF. The EIA system could detect as low as 1 pg/ml of NGF. NGF content was expressed as picograms of NGF per milligram of wet weight of sciatic nerve. NGF recov-
Clinical Observations
All diabetic rats showed marked impairment of growth. At the beginning of the experiment, the rats weighed 124 -t 11 g. Four weeks later, rats that had received citrate buffer alone weighed 348 rt: 27 g, whereas those given ST2 weighed only 165 f 51 g. Rats given STZ in combination with 4-MC weighed 186 + 53 g, indicating that 4-MC treatment did not have any effect on the body weight of diabetic rats. All diabetic rats showed marked h~erglycemia. 4MC treatment did not have any effect on glucose level (144 + 54 mg/dl in control rats, 607 -t 81 mg/dl in diabetic rats, and 650 +- 135 mg/dl in 4-MC-administered diabetic rats). Electrophysiological Examination
MNCVs at Weeks 0 and 2 were not si~i~cantly different among the three groups. At the end of Week 4, untreated diabetic rats showed no age-related increment of MNCV. The difference in MNCV between control rats (42.1 + 2.4 m/s) and untreated diabetic rats (34.9 + 3.6 m/s) after 4 weeks reached significance (P < 0.05, Fig. 1). 4-MC-administered rats showed an improved MNCV (40.8 & 3.2 m/s) and there was no significant difference between it and that of control rats. MNCVs of 4-MC-administered diabetic rats were significantly faster than those of untreated diabetic rats (P < 0.01). NGF Levels in the Sciatic Nerves
NGF levels at 4 weeks were significantly decreased to 0.15 1- 0.05 and 0.21 + 0.07 pg NGF/mg wet wt in the sciatic nerves of diabetic and 4-MC-administered dia-
294
HANAOKA
11 *** ii
I
0
2 Time
4
ET
AL.
reduction in myelin thickness. A recent study showed that myelin is more severely affected than the axon and that myelin thickness is reduced earlier than axon diameter in ST&induced diabetic rats (5). Diabetic rats show a variety of detrimental alterations in peripheral nerve such as accumulation of sorbitol pathway metabolites (23,24), depletion of myoinositol (25), reduced nerve blood flow, and hypoxia in the endoneurium (26). It is known that slow axonal transport of neurofilaments in peripheral nerves is reduced in STZ-induced diabetic rats (27). Accumulation of choline acetyltransferase activity proximal to a sciatic nerve crush was reduced in experimental diabetes in rats (28). Furthermore, several groups have found that diabetes causes alterations in fast and slow orthograde and retrograde transport in several classes of neurons (29-31). It was demonstrated that the retrograde axonal transport of ‘251-labeled NGF was reduced in experimental diabetic rats (6). A recent study has shown that endogenous levels of NGF in sciatic nerves of STZ-in-
(weeks)
FIG. 1. Effect of 4-MC on MNCV in the sciatic nerve. MNCVs of 4-MC-admi~stered diabetic rats (e, n = 11) were significantly faster than those of untreated diabetic rats (A, n = 9) (P .c O.Ol), but no significant difference was found between them and those of control rats (0, n = 7) at 4 weeks from the start of STZ injection. MNCVs of untreated diabetic rats were significantly slower than those of control rats (P < 0.05). The values represent the mean f SD. *P < 0.01; **P
