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Mechanical hyperalgesia in streptozotocin-diabetic rats is not sympathetically maintained
171
Brain Research, 616 (1993) 171-175 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 18987
Mechanical hyperalgesia in streptozotocin-diabetic rats is not sympathetically maintained Sara C. Ahlgren
a,d
and Jon D. Levine
a,b,c,d,e
Departments of a Anatomy, b Medicine, c Oral and Maxillofacial Surgery, d Biomedical Sciences Graduate Program and e Program in Neuroscience, University of California, San Francisco (USA) (Accepted 2 February 1993)
Key words." Streptozotocin-induced diabetes; Rat; Peripheral neuropathy; Sympathetically maintained pain; Norepinephrine; Mechanical nociceptive threshold
It has been suggested that the mechanism underlying the pain that occurs in patients with diabetic neuropathy may be similar to that mediating sympathetically maintained pain (SMP), such as occurs in patients with reflex sympathetic dystrophy. To evaluate this suggestion we have examined a model of diabetes mellitus, the streptozotocin-diabetic (STZ-D) rat, for features characteristic of SMP. We demonstrate that the decrease in nociceptive threshold observed in the STZ-D rat is not attenuated by chemical sympathectomy nor exacerbated by intradermal injection of norepinephrine (NE). In addition, the NE content is markedly decreased in the skin of the STZ-D rat. These results suggest that altered nociceptive thresholds associated with diabetic neuropathy are not sympathetically maintained.
INTRODUCTION Abnormal pain sensations, including paresthesias and dysesthesias occur in association with several metabolic, nutritional, toxic, and traumatic disorders. One of the most common disorders producing a painful peripheral neuropathy is diabetes mellitus. The abnormal pain sensations associated with diabetes may be acute and self-limiting2 or chronic 5'4z, and usually do not respond to available analgesic treatments 29. Sympathetically maintained pain 8'2° (SMP), is a pain syndrome associated with a variety of neuropathies which commonly follow traumatic injury of peripheral n e r v e s 8'9'16'36. SMP is characterized by the relief of pain and hyperalgesia after sympathetic block16'32'34 and the rekindling or exacerbation of pain after increased activity in the sympathetic postganglionic neuron or after administration of norepinephrine (NE) 3t'36. These features have also been observed experimentally after nerve trauma t3'23'24"26. The suggestion that painful diabetic neuropathy might be sympathetically maintained stems from the physiological observation that regenerating fibers are sensitive to epinephrine 24 and the
anatomical observation that there are actively regenerating fibers associated with diabetic neuropathy, and that these regenerating fibers may be more prevalent in those diabetic patients who suffer from painful neuropathy 6'7. To date, however, studies of the contribution of the sympathetic nervous system to the pain of diabetic neuropathy have been inconclusive3'43. In this study we have examined the role of the sympathetic nervous system in maintaining the mechanical hyperalgesia following streptozotocin-induced diabetes ( S T Z - D ) 1'11'14'39.
MATERIALS AND METHODS Animal model The experiments were performed on adult male Sprague-Dawley rats (220-350 g; Bantin and Kingman, Fremont, CA). Diabetes was induced by subcutaneous injection of streptozotocin (STZ; 70 mg/kg; Sigma, St. Louis, MO), a pancreatic fl-cell toxin 19'38. Controls (CON) were age-matched untreated rats. The onset of the diabetic state was assessed by the presence of glucosuria (urine glucose greater than 500 mg/dl; Ames Ketostix, Miles, Inc., Elkhardt, IN). Experiments were performed after the 30th day following induction of diabetes, a time when STZ-D rats demonstrate significant and stable mechanical hyperalgesia I.
Correspondence: J.D. Levine, Department of Anatomy, Box 0452A, University of California, San Francisco, CA 94143-0452, USA. Fax: (1) (415) 476-4845.
172 6 - O H D A treatment, and again on day 37, after 6 - O H D A treatment. Since diabetic rats have decreased mechanical nociceptive thresholds compared to control rats, the effect of the intradermally injected agents is expressed as the percentage change from the baseline prior to administration of the test agent, so that changes in the C O N and STZ rats could be compared. The d o s e - r e s p o n s e curves for NE were attained in a separate group of rats, by sequential intradermal injections of higher doses of test agent, in a volume of 2.5 /zl, with three m e a s u r e m e n t s per dose, at 5, 10, and 15 min post-injection, and each increasing dose given at 25-min intervals. NE and A23187 were co-injected with the same needle penetration, each in a volume of 2.5/zl separated by an air bubble in the syringe, with six measurements following the injection, at 5-min intervals.
Chemical sympathectomy To eliminate the effect of sympathetic postganglionic neuron terminals in the skin, 6-hydroxydopamine (6-OHDA) was injected intraperitoneally over a 7-day period ending 24 h before the study of hyperalgesia 2s. On days 1 and 2 of 6 - O H D A treatment, the rats received 50 m g / k g , and on days 3, 4, and 7 they received 100 m g / k g . To evaluate the relative completeness of the chemical sympathectomy procedure, subdermal tissue from the dorsal surface of the hind paws was harvested and assayed for its NE content using HPLC with coulometric analysis (ESA, Inc. Bedford, MA). To harvest tissue, rats were anesthetized and perfused with 0.1 M phosphate-buffered saline containing 0.27 m g / m l MgSO 4 chilled to 4°C. T h e n an 8-10 mg (wet weight) piece of subdermal tissue was excised from the dorsal surface of the hind paws and homogenized in 100x tissue volume of 0.001 N perchloric acid with 10 m M dithiothreitol. Each sample was then incubated with acid-washed alumina, and washed and aspirated through microfilters. The alumina was then washed with 200 tzl 0.001 N perchloric acid, 24 tzl of this wash was separated by HPLC and the NE content was determined by coulometric analysis.
Drugs The test agents used in this study were ( - ) - n o r e p i n e p h r i n e bitartrate (NE), the calcium ionophore A23187, and the postganglionic sympathetic neuron toxin 6-hydroxydopamine (6-OHDA) (all Sigma, St. Louis, MO). A23187 was dissolved in a 1:1 ethanol-saline stock solution, with further dilutions in saline (0.9% NaCI). NE was dissolved in saline. 6 - O H D A was dissolved in a saline vehicle with 1% ascorbic acid.
Mechanical nociceptit,e threshold The mechanical nociceptive threshold was quantified with an Ugo Basile Analgesymeter (Stoelting, Chicago, IL). This device generates a mechanical force that increases linearly with time. The force is applied directly over the site, marked by a felt tip pen, where injections will be placed, on the dorsum of the rats hind paw, by a dome-shaped plunger, (diameter 1.4 mm, radius of curvature 36°). The nociceptive threshold is defined as the force, in grams, at which the rat withdraws its paw. Rats were trained for 5 days prior to induction of diabetes. This training consists of repeated paw-withdrawal tests at 5-rain intervals, for 2 h a day. The average of the last six m e a s u r e m e n t s on the day STZ or vehicle was given is defined as the baseline nociceptive threshold. This training was repeated each testing day after injection of STZ or vehicle, and the last 6 m e a s u r e m e n t s constituted the threshold for that day. This training procedure reduces variability of the nociceptive threshold on repeated testing z7. In 8 STZ-D rats and 8 C O N rats, thresholds were evaluated again on day 30, prior to
Statistics Statistics were performed using Student's t-test or A N O V A , as appropriate. A P value less than 0.05 was considered significant.
RESULTS
Prior to injection of STZ, the mechanical nociceptive thresholds in STZ-D and CON groups of rats were not significantly different (t30 = -- 9.44; P > 0.05; inset Fig. 1). Thirty days following induction of diabetes, the STZ-D rats were significantly hyperalgesic compared to controls (t3(~= -6.58; P < 0.0001; Fig. 1), however,
STZ/6OHDA ~ STZ/CON
~
CON/ CON (n=10)
CON/ 60HDA (n=6)
CON/6OHDA CON/CON
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._
12o, 115, 110. 105,
-
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SIZt CON (n=8)
-35
-s . . . .
B ....
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lb . . . . Days
~ .... (post
2b . . . .
injection
2's . . . .
sb . . . .
.s~ . . . .
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o f STZ)
Fig. 1. The effect of 6-hydroxydopamine (6-OHDA) treatment on mechanical nociceptive threshold in the hind paws of streptozotocin-diabetic (STZ-D) and control (CON) rats. There was no significant change in thresholds in C O N rats when they were tested on day 30. However, there was a significant decrease in threshold in STZ-D rats when they were tested on day 30. Subsequent treatment with 6 - O H D A did not alter thresholds in C O N rats ( C O N / 6 - O H D A ) compared to vehicle-treated C O N rats ( C O N / C O N ) , nor in STZ-D ( S T Z / 6 - O H D A ) compared to vehicle-treated STZ-D rats ( S T Z - D / C O N ) . In this and subsequent figures data are presented as mean _+S.E.M. Inset: initial mechanical nociceptive threshold (g) in four groups of rats.
173 15
1200 -
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5 o
200.
.c_
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-25-30.
STZ NE (1 ~g) (n=12)
STZA23187 (1 lag) (n=6)
STZNE (11Jg) + A23187 (1 pg) (n= 14)
CON NE (1 #.g) + A23187 (ling) (n=6)
OSTZ-D/ 6-OHDA
STZ-D/ CON
CON/ 6-OHDA
CON/ CON
Fig. 2. Mean concentration of NE, expressed as pg N E / t i s s u e wet weight, in subdermal tissue collected after treatment for 7 days with 6 - O H D A or vehicle. * Differs from C O N / C O N , P < 0.05; Not significantly different from S T Z - D / C O N .
the nociceptive threshold in the S T Z - D subgroup, destined to receive 6 - O H D A ( S T Z / 6 - O H D A ; n = 8), was not significantly different from the threshold in the S T Z - D subgroup that was given vehicle at this time ( S T Z / C O N ; n = 8 ; t14 = - 0 . 5 5 ; P > 0.05). Similarly, on day 30 the mechanical thresholds of the subgroup of C O N rats that were to receive 6 - O H D A ( C O N / 6 O H D A ; n = 6) were not significantly different from the subgroups of C O N rats that were to receive vehicle ( C O N / C O N ; t14 = - 1 . 3 3 ; P > 0.05; n = 10), and the mechanical thresholds were not significantly different from those seen on day 0 (t15 = 1.9; P > 0.05). The injection of 6 - O H D A started on day 30, and continued over the next 7 days. On day 38 there was no change in the mechanical threshold of the rats that had received 6 - O H D A relative to their vehicle-receiving counterparts, for either C O N (tt4 = - 0 . 6 3 ; P > 0.05) or S T Z - D (t14 = 0.06; P > 0.05) rats (Fig. 1). At the end of the last day of testing, subcutaneous tissue was harvested from the dorsal surface of the hind paw, of the treated and non-treated rats, for analysis of N E content. Non-treated S T Z - D rats had significantly less N E than non-treated C O N rats (t 4 = 2.06; P > 0.05; Fig. 2). The 6 - O H D A treatment significantly decreased the N E concentration in C O N rats,
29
,51
0
.c_ ~ co) o r0
10-
-5qO -15-20 . . . . ~ 1
lb
1~0
NE (l~g)
Fig. 3. D o s e - r e s p o n s e curve for the effect of NE on mechanical nociceptive threshold. [ n , C O N rats, ( n = 12) and e, STZ-D rats (n = 12)].
Fig. 4. The percentage change in mechanical nociceptive threshold produced by N E (1 ~g); A23187 (1 # g ) and the combination of NE and A23187 in STZ-D rats; and for the combination in C O N rats. Neither NE nor A23187 alone resulted in significant changes in the mechanical thresholds in STZ-D rats, but the combination caused a significant decrease in mechanical thresholds in both STZ-D and C O N rats.
compared to untreated C O N rats (t 5 = 2.66, P < 0.05). However, 6 - O H D A did not significantly reduce the tissue content of N E in S T Z - D rats compared to the untreated S T Z - D rats (t 6 = 1.08; P > 0.05), presumably because the levels in the S T Z - D rats were already markedly reduced prior to 6 - O H D A administration. In separate groups of rats (STZ-D, n = 6; CON, n = 6), the effect of injection of N E into the skin on mechanical nociceptive threshold was tested. Intradermal injection of NE, in doses up to 100 /~g, did not significantly affect nociceptive threshold in either C O N ( F z = 0.144; P > 0.05) or S T Z - D ( F 2 = 0.84) rats ( P > 0.05; Fig. 3). However, the combination of 1 /zg N E with 1 /zg A23187 resulted in decreased mechanical threshold in both S T Z - D and C O N rats, with no significant difference in magnitude of this hyperalgesia (Fig. 4; t18 = 0.89; P > 0.05). As reported previously for normal control rats 15, A23187 (1 /xg), by itself, did not significantly affect mechanical nociceptive threshold in S T Z - D rats (t 5 = 0.47; P > 0.05). DISCUSSION In this study, we examined the hypothesis that the decreased mechanical nociceptive threshold seen in S T Z - D rats is, at least in part, sympathetically maintained. The hyperalgesia associated with STZ-induced diabetes was found not to be relieved by chemical sympathectomy. In fact, the tissue content of N E in the hind paw of the S T Z - D rat was greatly decreased at 38 days post-induction of diabetes, which also mitigates against the possibility that release of N E from endogenous stores drives the decrease in nociceptive thresholds. Consistent with our results, it has been shown that the content of the rate-limiting enzyme for N E synthesis, tyrosine hydroxylase, is decreased in sympathetic nerves
174 from 8-week S T Z - D rats, while the axon n u m b e r is not a l t e r e d , suggesting a deficit in c a t e c h o l a m i n e p r o d u c tion p r i o r to actual axon d e a t h 37. It has also b e e n d e m o n s t r a t e d that t h e r e a r e d e c r e a s e s at this time p o i n t in s u b s t a n c e P a n d calcitonin g e n e - r e l a t e d p e p tide in S T Z - D rats 1°. W h i l e it is possible t h a t t h e r e a r e N E - s e n s i t i v e axons or n o c i c e p t o r s 4, this is unlikely d u e to o u r o b s e r v a t i o n that e x o g e n o u s a d m i n i s t r a t i o n o f N E d i d not p r o d u c e an i n c r e a s e in hyperalgesia. In light o f this o b s e r v a t i o n , it is not surprising to n o t e that a n e c d o t a l clinical e v i d e n c e i n d i c a t e s t h a t it is r a r e to find reflex s y m p a t h e t i c d y s t r o p h y s y n d r o m e in p a t i e n t s with d i a b e t e s 25, even t h o u g h d i a b e t e s is an e x t r e m e l y c o m m o n disease. T h e r e have also b e e n m a n y clinical r e p o r t s t h a t suggest that d i a b e t e s is a s s o c i a t e d with a u t o n o m i c hypofunction, in p a t i e n t s b o t h with a n d without p a i n 21'41. F u r t h e r m o r e , t r e a t m e n t with a clonid i n e skin patch, which has b e e n r e p o r t e d to relieve p a i n in p a t i e n t s with S M P 9, was r e c e n t l y shown not to have a significant p a i n - r e l i e v i n g effect in p a t i e n t s suffering from p a i n f u l d i a b e t i c n e u r o p a t h y 43, c o n s i s t e n t with the suggestion that p a i n in d i a b e t e s is not s y m p a thetically m a i n t a i n e d , a l t h o u g h t h e s e a u t h o r s suggest the possibility t h a t t h e r e is a c l o n i d i n e - s e n s i t i v e subset of patients. N E a p p l i c a t i o n d i d not e n h a n c e m e c h a n i c a l hyperalgesia, unlike its effect on SMP. T h e r e f o r e , it a p p e a r s that the m e c h a n i c a l h y p e r a l g e s i a a s s o c i a t e d with diab e t e s is d i f f e r e n t from that i n d u c e d by d i r e c t t r a u m a to nerves in which sensitivity to s y m p a t h e t i c s t i m u l a t i o n and a p p l i c a t i o n o f N E is f o u n d 8'13'1s'23"26'4°, a l t h o u g h not all p a i n states following nerve t r a u m a a r e r e s p o n sive to s y m p a t h e t i c s t i m u l a t i o n o r r e l i e v e d by sympathectomy 12,33,34.T h e r e have also b e e n r e p o r t s o f N E d e p l e t i o n following p e r i p h e r a l nerve constriction 35. T h e h y p e r a l g e s i a of d i a b e t i c n e u r o p a t h y also a p p e a r s diff e r e n t from that o f nerve t e r m i n a l t r a u m a , such as c h l o r o f o r m t r e a t m e n t o f the h i n d p a w skin in the rat 15, a n d some forms of i n f l a m m a t o r y h y p e r a l g e s i a 17'22'3° which are sensitive to e x o g e n o u s N E . T h a t N E plus A23187 i n d u c e h y p e r a l g e s i a in t h e S T Z - D rat is consistent with t h e h y p o t h e s i s that functional N E r e c e p t o r s exist in the i n t r a d e r m a l s p a c e 15. W e cannot, from o u r e x p e r i m e n t , d e t e r m i n e which cell type c o n t a i n s the r e c e p t o r . T h e deficit in N E c o n t e n t of t h e skin of t h e S T Z - D rat that we have d e m o n s t r a t e d is c o n s i s t e n t with t h e d e c r e a s e in tyrosine hydroxylase c o n t e n t o f s y m p a t h e t i c nerves p r i o r to a c t u a l axon d e a t h which has b e e n shown by o t h e r s 37. W e have previously shown that c h e m i c a l s y m p a t h e c t o m y with 6 - O H D A , a toxin which is selectively t a k e n up by s y m p a t h e t i c t e r m i n a l s a n d results in d e p l e t i o n o f n e u r o t r a n s m i t t e r stores a n d nerve t e r m i n a l injury 28, r e m o v e s t h e ability o f N E a n d
A23187 to p r o d u c e h y p e r a l g e s i a ~5 in C O N rats, c o m p a t i b l e with the suggestion that in the skin the r e c e p t o r is on t h e s y m p a t h e t i c p o s t - g a n g l i o n i c n e u r o n t e r m i n a l . T h e i m p l i c a t i o n o f t h e s e o b s e r v a t i o n s to the c o n d i t i o n p r e s e n t in t h e S T Z - D rat is t h a t the S P G N t e r m i n a l is intact, a l t h o u g h the N E c o n t e n t is d e c r e a s e d . This is p r e s u m a b l y b e c a u s e the S T Z is p r o d u c i n g its d e p l e t i o n of N E stores via a m e c h a n i s m which leaves t h e nerve t e r m i n a l intact a n d responsive to N E . T h e s e results suggest t h a t d i a b e t i c h y p e r a l g e s i a , as r e p r e s e n t e d by the S T Z - D rat, is not s y m p a t h e t i c a l l y m a i n t a i n e d , a n d thus, d e s p i t e similarities, p r e s u m a b l y reflects a d i f f e r e n t u n d e r l y i n g m e c h a n i s m from t h a t of SMP. This work was supported by NIH Grant NS21647. We wish to thank Michael Gold and Paul Green for technical assistance, and Philip Heller for many helpful discussions during the preparation of the manuscript.
Acknowledgements.
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30 Tonussi, C.R. and Ferreira, S.H., Rat knee-joint carrageenin incapacitation test: an objective screen for central and peripheral analgesics, Pain, 48 (1992) 421-427. 31 Torebj6rk, E., Clinical and neurophysiological observations relating to pathophysiological mechanisms in reflex sympathetic dystrophy. In M. Stanton-Hicks, W. Janig and R.A. Boas (Eds.), Reflex Sympathetic Dystrophy, Kluwer, Boston, 1990, pp. 71-80. 32 Treede, R.-D., Davis, K.D., Campbell, J.N. and Raja, S.N., The plasticity of cutaneous hyperalgesia during sympathetic ganglion blockade in patients with neuropathic pain, Brain 115 (1992) 607-621. 33 Vallin, J.A. and Kingery, W.S., Adjacent neuropathic hyperalgesia in rats: a model for sympathetic independent pain, Neurosci. Lett., 133 (1991) 241-244. 34 Wahren, L.K., Torebj6rk, E. and Nystrom, B., Quantitative sensory testing before and after regional guanethidine block in patients with neuralgia in the hand, Pain, 46 (1991) 23-30. 35 Wakisaka, S., Kajander, K.C. and Bennett, G.J., Abnormal skin temperature and abnormal sympathetic vasomotor innervationin an experimental painful peripheral neuropathy, Pain, 46 (1991) 299-313. 36 Wallin, G., Torebj6rk, H. and Hallin, R., Preliminary observations on the pathophysiology of hyperalgesia in the causalgic pain syndrome. In Y. Zotterman (Ed.), Sensory Function in the Skin of Primates, Pergamon, Oxford, 1976, pp. 489-499. 37 Webster, G.J.M., Petch, E.W.A. and Cowen, T., Streptozotocininduced diabetes in rats causes neuronal deficits in tyrosine hydroxylase and 5-hydroxytryptamine specific to mesenteric perivascular sympathetic nerves and without loss of nerve fibers, Exp. Neurol., 113 (1991) 53-62. 38 Weiss, R.B., Streptozocin: a review of its pharmacology, efficacy and toxicity, Cancer Treat. Rep., 66 (1982) 427-437. 39 Wuarin-Bierman, L., Zahnd, G.R., Kaufman, F., Burklen, L. and Adler, J., Hyperalgesia in spontaneous and experimental animal models of diabetic neuropathy, Diabetologia, 30 (1987) 653-658. 40 Xie, Y. and Xiao, W., Electrophysiological evidence for hyperalgesia in the peripheral neuropathy, Sci. Chin. Ser. B, 33 (1990) 663-72. 41 Young, R.J., Zhou, Y.Q., Rodriguez, E., Prescott, R.J., Ewing, D.J. and Clarke, B.F., Variable relationship between peripheral somatic and autonomic neuropathy in patients with different syndromes of diabetic polyneuropathy, Diabetes, 35 (1986) 192197. 42 Young, R.J., Ewing, D.J. and Clarke, B.F., Chronic and remitting painful diabetic polyneuropathy, Diabetes Care, 11 (1988) 34-40. 43 Zeigler, D., Lynch, S.A., Muir, J., Benjamin, J. and Max, M.B., Transdermal clonidine versus placebo in painful diabetic neuropathy, Pain, 48 (1992) 403-408.
Brain Research, 616 (1993) 171-175 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
BRES 18987
Mechanical hyperalgesia in streptozotocin-diabetic rats is not sympathetically maintained Sara C. Ahlgren
a,d
and Jon D. Levine
a,b,c,d,e
Departments of a Anatomy, b Medicine, c Oral and Maxillofacial Surgery, d Biomedical Sciences Graduate Program and e Program in Neuroscience, University of California, San Francisco (USA) (Accepted 2 February 1993)
Key words." Streptozotocin-induced diabetes; Rat; Peripheral neuropathy; Sympathetically maintained pain; Norepinephrine; Mechanical nociceptive threshold
It has been suggested that the mechanism underlying the pain that occurs in patients with diabetic neuropathy may be similar to that mediating sympathetically maintained pain (SMP), such as occurs in patients with reflex sympathetic dystrophy. To evaluate this suggestion we have examined a model of diabetes mellitus, the streptozotocin-diabetic (STZ-D) rat, for features characteristic of SMP. We demonstrate that the decrease in nociceptive threshold observed in the STZ-D rat is not attenuated by chemical sympathectomy nor exacerbated by intradermal injection of norepinephrine (NE). In addition, the NE content is markedly decreased in the skin of the STZ-D rat. These results suggest that altered nociceptive thresholds associated with diabetic neuropathy are not sympathetically maintained.
INTRODUCTION Abnormal pain sensations, including paresthesias and dysesthesias occur in association with several metabolic, nutritional, toxic, and traumatic disorders. One of the most common disorders producing a painful peripheral neuropathy is diabetes mellitus. The abnormal pain sensations associated with diabetes may be acute and self-limiting2 or chronic 5'4z, and usually do not respond to available analgesic treatments 29. Sympathetically maintained pain 8'2° (SMP), is a pain syndrome associated with a variety of neuropathies which commonly follow traumatic injury of peripheral n e r v e s 8'9'16'36. SMP is characterized by the relief of pain and hyperalgesia after sympathetic block16'32'34 and the rekindling or exacerbation of pain after increased activity in the sympathetic postganglionic neuron or after administration of norepinephrine (NE) 3t'36. These features have also been observed experimentally after nerve trauma t3'23'24"26. The suggestion that painful diabetic neuropathy might be sympathetically maintained stems from the physiological observation that regenerating fibers are sensitive to epinephrine 24 and the
anatomical observation that there are actively regenerating fibers associated with diabetic neuropathy, and that these regenerating fibers may be more prevalent in those diabetic patients who suffer from painful neuropathy 6'7. To date, however, studies of the contribution of the sympathetic nervous system to the pain of diabetic neuropathy have been inconclusive3'43. In this study we have examined the role of the sympathetic nervous system in maintaining the mechanical hyperalgesia following streptozotocin-induced diabetes ( S T Z - D ) 1'11'14'39.
MATERIALS AND METHODS Animal model The experiments were performed on adult male Sprague-Dawley rats (220-350 g; Bantin and Kingman, Fremont, CA). Diabetes was induced by subcutaneous injection of streptozotocin (STZ; 70 mg/kg; Sigma, St. Louis, MO), a pancreatic fl-cell toxin 19'38. Controls (CON) were age-matched untreated rats. The onset of the diabetic state was assessed by the presence of glucosuria (urine glucose greater than 500 mg/dl; Ames Ketostix, Miles, Inc., Elkhardt, IN). Experiments were performed after the 30th day following induction of diabetes, a time when STZ-D rats demonstrate significant and stable mechanical hyperalgesia I.
Correspondence: J.D. Levine, Department of Anatomy, Box 0452A, University of California, San Francisco, CA 94143-0452, USA. Fax: (1) (415) 476-4845.
172 6 - O H D A treatment, and again on day 37, after 6 - O H D A treatment. Since diabetic rats have decreased mechanical nociceptive thresholds compared to control rats, the effect of the intradermally injected agents is expressed as the percentage change from the baseline prior to administration of the test agent, so that changes in the C O N and STZ rats could be compared. The d o s e - r e s p o n s e curves for NE were attained in a separate group of rats, by sequential intradermal injections of higher doses of test agent, in a volume of 2.5 /zl, with three m e a s u r e m e n t s per dose, at 5, 10, and 15 min post-injection, and each increasing dose given at 25-min intervals. NE and A23187 were co-injected with the same needle penetration, each in a volume of 2.5/zl separated by an air bubble in the syringe, with six measurements following the injection, at 5-min intervals.
Chemical sympathectomy To eliminate the effect of sympathetic postganglionic neuron terminals in the skin, 6-hydroxydopamine (6-OHDA) was injected intraperitoneally over a 7-day period ending 24 h before the study of hyperalgesia 2s. On days 1 and 2 of 6 - O H D A treatment, the rats received 50 m g / k g , and on days 3, 4, and 7 they received 100 m g / k g . To evaluate the relative completeness of the chemical sympathectomy procedure, subdermal tissue from the dorsal surface of the hind paws was harvested and assayed for its NE content using HPLC with coulometric analysis (ESA, Inc. Bedford, MA). To harvest tissue, rats were anesthetized and perfused with 0.1 M phosphate-buffered saline containing 0.27 m g / m l MgSO 4 chilled to 4°C. T h e n an 8-10 mg (wet weight) piece of subdermal tissue was excised from the dorsal surface of the hind paws and homogenized in 100x tissue volume of 0.001 N perchloric acid with 10 m M dithiothreitol. Each sample was then incubated with acid-washed alumina, and washed and aspirated through microfilters. The alumina was then washed with 200 tzl 0.001 N perchloric acid, 24 tzl of this wash was separated by HPLC and the NE content was determined by coulometric analysis.
Drugs The test agents used in this study were ( - ) - n o r e p i n e p h r i n e bitartrate (NE), the calcium ionophore A23187, and the postganglionic sympathetic neuron toxin 6-hydroxydopamine (6-OHDA) (all Sigma, St. Louis, MO). A23187 was dissolved in a 1:1 ethanol-saline stock solution, with further dilutions in saline (0.9% NaCI). NE was dissolved in saline. 6 - O H D A was dissolved in a saline vehicle with 1% ascorbic acid.
Mechanical nociceptit,e threshold The mechanical nociceptive threshold was quantified with an Ugo Basile Analgesymeter (Stoelting, Chicago, IL). This device generates a mechanical force that increases linearly with time. The force is applied directly over the site, marked by a felt tip pen, where injections will be placed, on the dorsum of the rats hind paw, by a dome-shaped plunger, (diameter 1.4 mm, radius of curvature 36°). The nociceptive threshold is defined as the force, in grams, at which the rat withdraws its paw. Rats were trained for 5 days prior to induction of diabetes. This training consists of repeated paw-withdrawal tests at 5-rain intervals, for 2 h a day. The average of the last six m e a s u r e m e n t s on the day STZ or vehicle was given is defined as the baseline nociceptive threshold. This training was repeated each testing day after injection of STZ or vehicle, and the last 6 m e a s u r e m e n t s constituted the threshold for that day. This training procedure reduces variability of the nociceptive threshold on repeated testing z7. In 8 STZ-D rats and 8 C O N rats, thresholds were evaluated again on day 30, prior to
Statistics Statistics were performed using Student's t-test or A N O V A , as appropriate. A P value less than 0.05 was considered significant.
RESULTS
Prior to injection of STZ, the mechanical nociceptive thresholds in STZ-D and CON groups of rats were not significantly different (t30 = -- 9.44; P > 0.05; inset Fig. 1). Thirty days following induction of diabetes, the STZ-D rats were significantly hyperalgesic compared to controls (t3(~= -6.58; P < 0.0001; Fig. 1), however,
STZ/6OHDA ~ STZ/CON
~
CON/ CON (n=10)
CON/ 60HDA (n=6)
CON/6OHDA CON/CON
5-
0-
-5-
-a O
d-
-10-
-15© (3) -20 c o dO -25-
_~
14o
._
12o, 115, 110. 105,
-
100
STZ/ 6-OHDA (n=8)
-30~'
SIZt CON (n=8)
-35
-s . . . .
B ....
~ ....
lb . . . . Days
~ .... (post
2b . . . .
injection
2's . . . .
sb . . . .
.s~ . . . .
4b
o f STZ)
Fig. 1. The effect of 6-hydroxydopamine (6-OHDA) treatment on mechanical nociceptive threshold in the hind paws of streptozotocin-diabetic (STZ-D) and control (CON) rats. There was no significant change in thresholds in C O N rats when they were tested on day 30. However, there was a significant decrease in threshold in STZ-D rats when they were tested on day 30. Subsequent treatment with 6 - O H D A did not alter thresholds in C O N rats ( C O N / 6 - O H D A ) compared to vehicle-treated C O N rats ( C O N / C O N ) , nor in STZ-D ( S T Z / 6 - O H D A ) compared to vehicle-treated STZ-D rats ( S T Z - D / C O N ) . In this and subsequent figures data are presented as mean _+S.E.M. Inset: initial mechanical nociceptive threshold (g) in four groups of rats.
173 15
1200 -
v lo!
~ 7000.~
•a o r-
©
~~
5 o
200.
.c_
-io -15 -20 -
0r-
-25-30.
STZ NE (1 ~g) (n=12)
STZA23187 (1 lag) (n=6)
STZNE (11Jg) + A23187 (1 pg) (n= 14)
CON NE (1 #.g) + A23187 (ling) (n=6)
OSTZ-D/ 6-OHDA
STZ-D/ CON
CON/ 6-OHDA
CON/ CON
Fig. 2. Mean concentration of NE, expressed as pg N E / t i s s u e wet weight, in subdermal tissue collected after treatment for 7 days with 6 - O H D A or vehicle. * Differs from C O N / C O N , P < 0.05; Not significantly different from S T Z - D / C O N .
the nociceptive threshold in the S T Z - D subgroup, destined to receive 6 - O H D A ( S T Z / 6 - O H D A ; n = 8), was not significantly different from the threshold in the S T Z - D subgroup that was given vehicle at this time ( S T Z / C O N ; n = 8 ; t14 = - 0 . 5 5 ; P > 0.05). Similarly, on day 30 the mechanical thresholds of the subgroup of C O N rats that were to receive 6 - O H D A ( C O N / 6 O H D A ; n = 6) were not significantly different from the subgroups of C O N rats that were to receive vehicle ( C O N / C O N ; t14 = - 1 . 3 3 ; P > 0.05; n = 10), and the mechanical thresholds were not significantly different from those seen on day 0 (t15 = 1.9; P > 0.05). The injection of 6 - O H D A started on day 30, and continued over the next 7 days. On day 38 there was no change in the mechanical threshold of the rats that had received 6 - O H D A relative to their vehicle-receiving counterparts, for either C O N (tt4 = - 0 . 6 3 ; P > 0.05) or S T Z - D (t14 = 0.06; P > 0.05) rats (Fig. 1). At the end of the last day of testing, subcutaneous tissue was harvested from the dorsal surface of the hind paw, of the treated and non-treated rats, for analysis of N E content. Non-treated S T Z - D rats had significantly less N E than non-treated C O N rats (t 4 = 2.06; P > 0.05; Fig. 2). The 6 - O H D A treatment significantly decreased the N E concentration in C O N rats,
29
,51
0
.c_ ~ co) o r0
10-
-5qO -15-20 . . . . ~ 1
lb
1~0
NE (l~g)
Fig. 3. D o s e - r e s p o n s e curve for the effect of NE on mechanical nociceptive threshold. [ n , C O N rats, ( n = 12) and e, STZ-D rats (n = 12)].
Fig. 4. The percentage change in mechanical nociceptive threshold produced by N E (1 ~g); A23187 (1 # g ) and the combination of NE and A23187 in STZ-D rats; and for the combination in C O N rats. Neither NE nor A23187 alone resulted in significant changes in the mechanical thresholds in STZ-D rats, but the combination caused a significant decrease in mechanical thresholds in both STZ-D and C O N rats.
compared to untreated C O N rats (t 5 = 2.66, P < 0.05). However, 6 - O H D A did not significantly reduce the tissue content of N E in S T Z - D rats compared to the untreated S T Z - D rats (t 6 = 1.08; P > 0.05), presumably because the levels in the S T Z - D rats were already markedly reduced prior to 6 - O H D A administration. In separate groups of rats (STZ-D, n = 6; CON, n = 6), the effect of injection of N E into the skin on mechanical nociceptive threshold was tested. Intradermal injection of NE, in doses up to 100 /~g, did not significantly affect nociceptive threshold in either C O N ( F z = 0.144; P > 0.05) or S T Z - D ( F 2 = 0.84) rats ( P > 0.05; Fig. 3). However, the combination of 1 /zg N E with 1 /zg A23187 resulted in decreased mechanical threshold in both S T Z - D and C O N rats, with no significant difference in magnitude of this hyperalgesia (Fig. 4; t18 = 0.89; P > 0.05). As reported previously for normal control rats 15, A23187 (1 /xg), by itself, did not significantly affect mechanical nociceptive threshold in S T Z - D rats (t 5 = 0.47; P > 0.05). DISCUSSION In this study, we examined the hypothesis that the decreased mechanical nociceptive threshold seen in S T Z - D rats is, at least in part, sympathetically maintained. The hyperalgesia associated with STZ-induced diabetes was found not to be relieved by chemical sympathectomy. In fact, the tissue content of N E in the hind paw of the S T Z - D rat was greatly decreased at 38 days post-induction of diabetes, which also mitigates against the possibility that release of N E from endogenous stores drives the decrease in nociceptive thresholds. Consistent with our results, it has been shown that the content of the rate-limiting enzyme for N E synthesis, tyrosine hydroxylase, is decreased in sympathetic nerves
174 from 8-week S T Z - D rats, while the axon n u m b e r is not a l t e r e d , suggesting a deficit in c a t e c h o l a m i n e p r o d u c tion p r i o r to actual axon d e a t h 37. It has also b e e n d e m o n s t r a t e d that t h e r e a r e d e c r e a s e s at this time p o i n t in s u b s t a n c e P a n d calcitonin g e n e - r e l a t e d p e p tide in S T Z - D rats 1°. W h i l e it is possible t h a t t h e r e a r e N E - s e n s i t i v e axons or n o c i c e p t o r s 4, this is unlikely d u e to o u r o b s e r v a t i o n that e x o g e n o u s a d m i n i s t r a t i o n o f N E d i d not p r o d u c e an i n c r e a s e in hyperalgesia. In light o f this o b s e r v a t i o n , it is not surprising to n o t e that a n e c d o t a l clinical e v i d e n c e i n d i c a t e s t h a t it is r a r e to find reflex s y m p a t h e t i c d y s t r o p h y s y n d r o m e in p a t i e n t s with d i a b e t e s 25, even t h o u g h d i a b e t e s is an e x t r e m e l y c o m m o n disease. T h e r e have also b e e n m a n y clinical r e p o r t s t h a t suggest that d i a b e t e s is a s s o c i a t e d with a u t o n o m i c hypofunction, in p a t i e n t s b o t h with a n d without p a i n 21'41. F u r t h e r m o r e , t r e a t m e n t with a clonid i n e skin patch, which has b e e n r e p o r t e d to relieve p a i n in p a t i e n t s with S M P 9, was r e c e n t l y shown not to have a significant p a i n - r e l i e v i n g effect in p a t i e n t s suffering from p a i n f u l d i a b e t i c n e u r o p a t h y 43, c o n s i s t e n t with the suggestion that p a i n in d i a b e t e s is not s y m p a thetically m a i n t a i n e d , a l t h o u g h t h e s e a u t h o r s suggest the possibility t h a t t h e r e is a c l o n i d i n e - s e n s i t i v e subset of patients. N E a p p l i c a t i o n d i d not e n h a n c e m e c h a n i c a l hyperalgesia, unlike its effect on SMP. T h e r e f o r e , it a p p e a r s that the m e c h a n i c a l h y p e r a l g e s i a a s s o c i a t e d with diab e t e s is d i f f e r e n t from that i n d u c e d by d i r e c t t r a u m a to nerves in which sensitivity to s y m p a t h e t i c s t i m u l a t i o n and a p p l i c a t i o n o f N E is f o u n d 8'13'1s'23"26'4°, a l t h o u g h not all p a i n states following nerve t r a u m a a r e r e s p o n sive to s y m p a t h e t i c s t i m u l a t i o n o r r e l i e v e d by sympathectomy 12,33,34.T h e r e have also b e e n r e p o r t s o f N E d e p l e t i o n following p e r i p h e r a l nerve constriction 35. T h e h y p e r a l g e s i a of d i a b e t i c n e u r o p a t h y also a p p e a r s diff e r e n t from that o f nerve t e r m i n a l t r a u m a , such as c h l o r o f o r m t r e a t m e n t o f the h i n d p a w skin in the rat 15, a n d some forms of i n f l a m m a t o r y h y p e r a l g e s i a 17'22'3° which are sensitive to e x o g e n o u s N E . T h a t N E plus A23187 i n d u c e h y p e r a l g e s i a in t h e S T Z - D rat is consistent with t h e h y p o t h e s i s that functional N E r e c e p t o r s exist in the i n t r a d e r m a l s p a c e 15. W e cannot, from o u r e x p e r i m e n t , d e t e r m i n e which cell type c o n t a i n s the r e c e p t o r . T h e deficit in N E c o n t e n t of t h e skin of t h e S T Z - D rat that we have d e m o n s t r a t e d is c o n s i s t e n t with t h e d e c r e a s e in tyrosine hydroxylase c o n t e n t o f s y m p a t h e t i c nerves p r i o r to a c t u a l axon d e a t h which has b e e n shown by o t h e r s 37. W e have previously shown that c h e m i c a l s y m p a t h e c t o m y with 6 - O H D A , a toxin which is selectively t a k e n up by s y m p a t h e t i c t e r m i n a l s a n d results in d e p l e t i o n o f n e u r o t r a n s m i t t e r stores a n d nerve t e r m i n a l injury 28, r e m o v e s t h e ability o f N E a n d
A23187 to p r o d u c e h y p e r a l g e s i a ~5 in C O N rats, c o m p a t i b l e with the suggestion that in the skin the r e c e p t o r is on t h e s y m p a t h e t i c p o s t - g a n g l i o n i c n e u r o n t e r m i n a l . T h e i m p l i c a t i o n o f t h e s e o b s e r v a t i o n s to the c o n d i t i o n p r e s e n t in t h e S T Z - D rat is t h a t the S P G N t e r m i n a l is intact, a l t h o u g h the N E c o n t e n t is d e c r e a s e d . This is p r e s u m a b l y b e c a u s e the S T Z is p r o d u c i n g its d e p l e t i o n of N E stores via a m e c h a n i s m which leaves t h e nerve t e r m i n a l intact a n d responsive to N E . T h e s e results suggest t h a t d i a b e t i c h y p e r a l g e s i a , as r e p r e s e n t e d by the S T Z - D rat, is not s y m p a t h e t i c a l l y m a i n t a i n e d , a n d thus, d e s p i t e similarities, p r e s u m a b l y reflects a d i f f e r e n t u n d e r l y i n g m e c h a n i s m from t h a t of SMP. This work was supported by NIH Grant NS21647. We wish to thank Michael Gold and Paul Green for technical assistance, and Philip Heller for many helpful discussions during the preparation of the manuscript.
Acknowledgements.
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