- Email: [email protected]
Dimethylsulfoxide (DMSO) blocks conduction in peripheral nerve C fibers: a possible mechanism of analgesia
Neuroscience Letters, 150 (1993) 145-148
145
Elsevier Scientific Publishers Ireland Ltd.
NSL 09283
Dimethylsulfoxide (DMSO) blocks conduction in peripheral nerve C fibers: a possible mechanism of analgesia M. Steven Evans a, K e n n e t h H. R e i d b a n d James B. Sharp Jr. c aDivision of Neurology, Department of Internal Medicine, Southern Illinois University School of Medicine, Springfield, IL (USA,), bDepartment of Anatomy and Neurobiology and CDepartment of Veterinary Medicine, University of Louisville School of Medicine, Louisville, K Y (USA) (Received 29 June 1992; Revised version received 3 November 1992; Accepted 5 November 1992)
Key words: Dimethylsulfoxide; Analgesia; Pain; C fiber; Nerve block Dimethylsulfoxide (DMSO) is readily absorbed through skin, and relieves musculoskeletal pain when applied topically to painful areas. We studied the effects of DMSO on C-type nerve fibers, which mediate pain sensation. DMSO was applied directly to exposed cat sural nerves. C fiber conduction velocity was slowed by DMSO, even in low concentrations (5-7% v/v). Higher concentrations completely blocked C fiber conduction, with a minimum blocking concentration of 9%. Onset of nerve block was almost immediate with 15% DMSO or higher concentrations. C fiber blockade may account for analgesia with DMSO.
Dimethylsulfoxide (DMSO) is a solvent with local analgesic properties. It penetrates skin quickly [11], so for analgesia it is applied directly to skin over the painful area. Initial reports on DMSO emphasized its usefulness as a local analgesic [10], and it became a popular remedy for relief from the musculoskeletal pain of arthritis, sprains and strains [13], even though never approved by the Food and Drug Administration for this use [7]. Clinical studies of DMSO found it to be analgesic [2, 4, 12, 14], although some studies found no benefit [16]. Somatic pain is mediated by unmyelinated C type nerve fibers terminating in the skin, muscle and joint capsules [5]. We have studied a possible mechanism of DMSO-induced analgesia by observing the effects of DMSO on conduction in C fibers of cat cutaneous nerves. (Some of these results were presented previously in abstract form [17].) Ninety percent DMSO in water (Eqigisic, Burlington Bio-Medical Co.) was diluted to the desired concentration (expressed as v/v) with deionized water and Hank's Balanced Salt Solution concentrate (Grand Island Biological Company). The final concentrations were (in mM) NaC1 136.9, glucose 11.0, N a H C O 3 15.1, KCI 5.4, CaCI2 1.8, MgSOa.7H20 0.8, KH2PO 4 0.7 and NaH2PO 42H20 0.6. The solute concentration was 280 mM, but total osmolarity was greater due to the DMSO (1588 Correspondence: M.S. Evans, Box 19230, Division of Neurology, Southern Illinois University School of Medicine, Springfield, IL 62794, USA. Fax: (1) (217) 788-5567.
mosmol for a 10% DMSO solution). If necessary, the solution was titrated to pH 7.4 with small amounts of NaH2CO3. Sural nerves of twenty-five random source 2-4 kg cats were studied. Cats were anesthetized with 40 mg/kg sodium pentobarbital intravenously, supplemented as needed to maintain a level of anesthesia sufficient to eliminate withdrawal reflexes. The left sural nerve and its epineurial sheath were carefully microdissected from the underlying muscle and fascia, but left in situ. Proximally, the sural nerve remained connected to the sciatic nerve, and distally remained covered by fascia overlying the gastrocnemius muscle. The nerve's blood supply was left intact. The nerve was placed in a small bath, a slotted glass cylinder with an internal length of 2.2 cm. Bathing medium was injected into the nerve bath manually with a syringe, and removed at a rate of 0.08 ml/min with an infusion pump. The volume of the nerve bath was 0.4 ml, so that a complete change of solution took 5 minutes. A bipolar stimulating electrode was placed on the nerve proximal to the bath, and a bipolar recording electrode distal to it (antidromic stimulation). The cat limb, electrodes and bath were then enclosed in a plastic tent kept at 100% humidity, 36°C. Stimulating and recording electrodes were fabricated from chlorided silver wires. Constant-voltage capacitance-isolated square wave stimuli, 0.5 ms duration, were given at 0.3-3 Hz. Stimulus intensity was set to twice the voltage at C fiber threshold, which ranged from 5 to 10 V
146
A
B
C
2O
10
0
'i
'
I
i
IN 6
~
60
80
°I .m--o, i 20
40
i
i
100
120
, w ~ w i i :
140
i
160
i
i
180 2 0 0
Min~
Fig. 1. A: sural nerve responses are stable. Top: C fiber waveform at beginning of experiment. A fiber responses at the beginning of the record are distorted because of the high gain (which saturates the signal averager) and slow timebase needed to demonstrate C responses. Positiveis upward in all traces. Middle: response 9 h later, with no DMSO treatment. Bottom: same nerve, C fiber response blocked 10 min after application of 10% DMSO. B: DMSO transiently increases spontaneous firing. Top: control sural nerve C fiber response. Middle: 1 min after 5% DMSO, background noise is dramatically increased by spontaneous action potential firing. Bottom: after 20 min in 5% DMSO, spontaneous firing is not apparent. C: DMSO blockade is reversible. A nerve was exposed to DMSO during the times indicated by dotted lines. Control responses (0-20 min) varied in amplitude. 30% and 40% DMSO quickly abolished C fiber responses, with the blockades reversed by washing. In this example, 50% DMSO caused irreversible blockade. (mean of 7.5 V + 0.37 S.E.M.). C fiber responses were identified by their slower conduction velocity, smaller amplitude and higher stimulus threshold compared to A fibers. Conventional electronic devices were used for recording and displaying responses. A general-purpose microcomputer averaged 16 responses, which were then plotted with a chart recorder. The gain and timebase needed to display C responses were incompatible with simultaneously recording A fiber responses. C fiber compound action potential amplitude (most positive point minus most negative point) and latency (to most positive point) were measured. In agreement with previous investigators [5, 6], we found that conduction velocity was stable, but C fiber response amplitudes and shapes varied from minute to minute. Because of this, we used complete block of C fiber responses as an endpoint (Fig. 1A), since this never occurred normally. Complete block was defined as loss of any organized trace of the original waveform. The sural nerve preparation was stable in long-term experiments. Two nerves were followed without drug treatment for nine hours, with no systematic changes in response amplitude or latency (Fig. 1A). During experiments, the health of the nerve was assessed by stroking hairs on the foot, causing action potentials to fire. In no case was responsiveness to this natural stimulus diminished, even after complete blockade of nerve conduction through the bath. Experiments were performed by increasing the D M S O concentration until C fibers were blocked, then lowering the concentration until the block reversed. Immediately after D M S O application, a 1-5 min transient period of increased noise was always seen with concentrations of
5% or more (Fig. 1B). This was due to asynchronous spontaneous firing of axons, transiently reducing the amplitude of evoked C fiber responses. D M S O effects on C fiber response amplitudes were concentration-dependent. Five percent and 7% D M S O tended to reduce C fiber response amplitudes, but the minimum blocking concentration was 9% (Figs. 1A, 2B, and C). In 14 experiments using 9% or 10% D M S O , C fiber responses were invariably reduced, and were completely blocked in 9 experiments (Figs. 1A and 2A). In these 9 experiments, blockade occurred 22.0 min _+ 2.0 after D M S O application. D M S O concentrations of 15% or more reliably blocked C fibers. All nerves exposed to 15% or more D M S O were quickly blocked (mean 6.3 minutes _+ 0.3, n = 15 experiments) (Fig. 2B). Five percent D M S O relieved block caused by higher concentrations (Fig. 2A), but reversal of established blockade was never seen with 9% D M S O or higher concentrations. In 11 experiments (8 nerves), C fiber blockade was produced by 9% to 20% D M S O , followed by application of 5%; recovery was good in l0 of the l l experiments. Because blockade was usually reversible, two or three experiments were performed on some nerves that recovered to 75% of their maximal p r e - D M S O response amplitude. Six nerves were exposed to low concentrations of D M S O for several hours (not illustrated). With such prolonged exposure, 1% to 7% D M S O blocked four nerves 6.25-8 h after application of drug, suggesting that long exposure to low concentrations of D M S O can reduce C fiber transmission. In three of the four nerves, blockade reversed after extended washing.
147
A
B "rime w~ Response
C
Amplimcle
Conce,;U~;on re, Amplitude
120
14 6
.~100 ,o
R
r
t
~
~¢0 60
10% DMS0
:.B ¢= 4o
lS
~ ,o
20
Oo
15% DMSO
2:
qT~,.~~ . . . . . . 1"0 " 20
30
40
50
60
5
70
10
3 15
Minutes
D
E
Time vs. Latency
F
Concentncion vs. latency 4
220
ii,.o[
1BO
160 14(1
RO~
120
7%
-'
100
7% []VI~
0
,'o 2'0 ;
,'o
MintCes
,oo[ go 7o
0'
5'
Con(:entz'e~n [% v/v]
1'o
Fig. 2. DMSO reduces C fiber response amplitude and conduction velocity. A, top: response in 5% DMSO. A, middle: 10% DMSO blocked C fiber responses. A, bottom: two minutes after reapplication of 5% DMSO, response returns. B: time-response curves for DMSO are shown. Changes are expressed as percentage of the mean amplitude of the 3-5 measurments immediately preceding DMSO application. Seven percent and lower concentrations did not block over 3-4 h. The minimum blocking concentration of DMSO was approximately 9%. Most nerves were blocked by a 55 min application of 9% DMSO, and drug application was not continued past that time. Concentrations of 15% and more blocked C responses quickly. Bars indicate standard error of the mean. For clarity, bars are not shown for 0% and 9% DMSO at times less than 35 min. For other observations, if bars are not shown, standard error of the mean is less than symbol size. Small numerals indicate number of experiments. C: Concentration-response curve f6r DMSO. Responses measured 35 min after drug application. Five percent and 7% DMSO slightly reduced response amplitudes, but the difference between these and the control values was not statistically significant (two-tailed t-test). Nine percent DMSO reduced C responses by 50% after 35 min, and eventually blocked most nerves. The difference between 9% DMSO and control is significant (P < 0.05). D: 7% DMSO increased C fiber response latency by 43% over control in this nerve (control latency 78 ms, 7% DMSO 112 ms). E: response latency did not change with time. F: increase in latency is related to DMSO concentration, but not to duration of DMSO exposure. Five percent to 10% DMSO all caused significant increases in response latency over control (P < 0.01). There was no difference in the latencies at 5 min compared to 35 min of DMSO exposure. D M S O r e d u c e d C fiber c o n d u c t i o n velocity (Fig. 2 D F). C o n c e n t r a t i o n s as low as 2% D M S O p r o d u c e d definite increases in r e s p o n s e latency. U n l i k e the effect on r e s p o n s e a m p l i t u d e , w h i c h t o o k tens o f m i n u t e s to dev e l o p with 9% D M S O , the effect o n r e s p o n s e l a t e n c y was a l m o s t i n s t a n t a n e o u s . This effect was stable d u r i n g the time o f d r u g exposure, a n d reversed after d r u g r e m o v a l . R e s p o n s e l a t e n c y i n c r e a s e d b y a l m o s t 200% with 10% D M S O , i m p l y i n g t h a t overall c o n d u c t i o n velocity in the nerve s e g m e n t b e t w e e n s t i m u l a t i n g a n d r e c o r d i n g elect r o d e s was r e d u c e d b y 50%. H o w e v e r , this represents a c o n s e r v a t i v e e s t i m a t e o f the effects o f D M S O , since the nerve s e g m e n t studied, u s u a l l y 5 c m long, was e x p o s e d to D M S O o n l y within the 2.2 c m l o n g nerve b a t h . T h e m e c h a n i s m o f the c o n d u c t i o n b l o c k i n d u c e d b y
D M S O m a y involve p o t a s s i u m c h a n n e l b l o c k a d e . S a w a d a a n d S a t o [18] f o u n d t h a t D M S O c a u s e d a r a p i d m e m b r a n e d e p o l a r i z a t i o n a n d decrease in m e m b r a n e c o n d u c t a n c e o f Aplysia n e u r o n s , c o n s i s t e n t with b l o c k a d e o f leak p o t a s s i u m channels. D M S O s l o w e d a c t i o n potential repolarization, and voltage clamp study s h o w e d t h a t D M S O i n h i b i t e d the d e l a y e d rectifier p o t a s sium channel. T h e i r s t u d y implies t h a t D M S O can p r o duce a d e p o l a r i z i n g nerve b l o c k t h r o u g h i n h i b i t i o n o f b o t h voltage-sensitive a n d voltage-insensitive p o t a s s i u m channels. D e p o l a r i z i n g b l o c k is consistent with results o f the p r e s e n t experiments, in which the t r a n s i e n t increase in electrical noise seen i m m e d i a t e l y after a p p l i c a t i o n o f D M S O suggests t h a t fibers have d e p o l a r i z e d sufficiently to allow s p o n t a n e o u s a c t i o n p o t e n t i a l firing. H o w
148 D M S O m i g h t specifically affect p o t a s s i u m channels is not clear, b u t M a y e r a n d A v i - D o r [15] have suggested that D M S O can c h a n g e the state o f h y d r a t i o n o f the p o tassium ion, which m i g h t alter its ability to p e n e t r a t e ion channels. The m e c h a n i s m o f D M S O ' s ability to p r o f o u n d l y reduce C fiber c o n d u c t i o n velocity is unclear. This effect is fast, stable a n d reversible. It is d e t e c t a b l e with low conc e n t r a t i o n s o f the drug. C o n d u c t i o n velocity r e d u c t i o n m a y also be caused by effects on p o t a s s i u m channels. One possibility is that persistent m e m b r a n e d e p o l a r i z a tion c o u l d facilitate s o d i u m channel i n a c t i v a t i o n , with c o n s e q u e n t slowing o f a c t i o n p o t e n t i a l generation. A seco n d possibility is t h a t an increase in m e m b r a n e resistance due to b l o c k a d e o f p o t a s s i u m channels m i g h t affect cond u c t i o n velocity, b u t in this case the m a g n i t u d e a n d direction o f the effect are n o t easily p r e d i c t e d [9]. C o n d u c tion velocity c o u l d also be r e d u c e d by increasing extracellular electrical resistance [8], b u t u n d e r n o r m a l conditions, c o n d u c t i o n velocity changes a p p r e c i a b l y only with m a n y f o l d increases in extracellular resistance. T h e ability o f 9% D M S O to b l o c k C fibers c o n t r a s t s with previous studies on m y e l i n a t e d A type nerve fibers, in which m u c h larger c o n c e n t r a t i o n s were needed. D a v i s et al. [3] f o u n d t h a t 50% D M S O was necessary to b l o c k muscle twitch in a frog sciatic n e r v e - g a s t r o c n e m i u s muscle p r e p a r a t i o n . Becker [1] f o u n d that 75% D M S O b l o c k e d b o t h A a n d C fibers. Shealey [19] studied a p r o longed small fiber a f t e r d i s c h a r g e in spinal cord, m e d u l l a a n d t e g m e n t u m following s t i m u l a t i o n o f the superficial radial a n d sural nerves, a n d f o u n d it to be b l o c k e d by e x p o s i n g the p e r i p h e r a l nerve to 5-10% D M S O , a result that c o u l d be d u e to b l o c k a d e o f C fibers. T h e present e x p e r i m e n t s suggest t h a t D M S O can be an effective topical analgesic. I f analgesia with D M S O d e p e n d s on b l o c k a d e o f C fibers, one r e q u i r e m e n t is t h a t the d r u g c o n c e n t r a t i o n a r o u n d C fibers leading f r o m the painful a r e a be b r o u g h t to 10% o r m o r e for several minutes. It is likely t h a t this s i t u a t i o n occurs clinically, since D M S O p e n e t r a t e s skin easily, a n d topical a p p l i c a t i o n o f 70 90% is c o m m o n l y used. I n s o f a r as D M S O selectively affects s o m a t i c C fibers, a selective a n a l g e s i a a n d loss o f t e m p e r a t u r e sensation, r a t h e r t h a n c o m p l e t e anesthesia, m a y be expected. L i g h t l y - m y e l i n a t e d A - d e l t a fibers t h a t m e d i a t e s h a r p (first) p a i n were n o t investigated in this study, but if unaffected b y D M S O , w o u l d allow s h a r p p a i n to persist, a n d deep, aching p a i n to be abolished. Deep, aching p a i n is p r e s e n t in arthritis a n d m u s c u loskeletal injuries, the c o n d i t i o n s for which D M S O has often been used.
This w o r k is p a r t o f a thesis s u b m i t t e d by M.S.E. to the G r a d u a t e School o f the U n i v e r s i t y o f Louisville, A u gust, 1984. T h e a u t h o r s t h a n k Jennifer E v a n s a n d D e a n N a r i t o k u for critical r e a d i n g o f the m a n u s c r i p t . 1 Becker, D.P., Young, H.F., Nulsen, F.E. and Jane, J.A., Physiological effects of dimethylsulfoxide on peripheral nerves: possible role in pain relief, Exp. Neurol., 24 (1969) 272-276. 2 Brown, J.H., DMSO its efficacy in acute musculoskeletal problems as evaluated by a 'double blind' study, Industrial Medicine and Surgery, 35 (1967) 777-781. 3 Davis, H.L., Davis, N.L. and Clemons, A.L., Procoagulant and nerve-blocking effects of DMSO, Ann. N.Y. Acad. Sci., 141 (1967) 310 325. 4 Demos, C.H., Beckloff, G.L., Donin, M.N. and Oliver, EM., Dimethylsulfoxide in musculoskeletal disorders, Ann. N.Y. Acad. Sci., 141 (1967)517 523. 5 Douglas, W.W. and Ritchie, J.M., Mammalian non-myelinated nerve fibers, Physiol. Rev., 42 (1962) 297-334. 6 Gasser, H.S., Unmedullated fibers originating in dorsal root ganglia, J. Gen. Physiol., 33 (1950) 650-690. 7 Harter, J.G., The status of dimethylsulfoxide from the perspective of the food and drug administration, Ann. N.Y. Acad. Sci., 411 (1983) 1-5. 8 Hodgkin, A i . , The relation between conduction velocity and the electrical resistance outside a nerve fiber, J. Physiol., 94 (1939) 560570. 9 Hunter, EJ., McNaughton, RA. and Noble, D., Analytical models of propagation in excitable cells, Prog. Biophys., 30 (1975) 99-144. 10 Jacob, S.W., Bischel, M. and Herschler, R.J., Dimethylsulfoxide (DMSO): a new concept in pharmacotherapy, Current Ther. Res., 6 (1964) 134-135. 11 Kolb, K.H., Jaenicke, G., Kramer, M. and Schulze, RE., Absorption, distribution and elimination of labeled dimethylsulfoxide in man and animals, Ann. N.Y. Acad. Sci., 141 (1967) 85-95. 12 Lockie, L.M. and Norcross, B.M., A clinical study on the effects of dimethylsulfoxide in 103 patients with acute and chronic musculoskeletal injuries and inflammations, Ann. N.Y. Acad. Sci., 141 (1967) 599-602. 13 MacGrady, R, The Persecuted Drug: The Story of DMSO, Doubleday and Company, Garden City, New York, 1973, 335 pp. 14 Matsumoto. J., Clinical trials of dimethylsulfoxide in rheumatoid arthritis patients in Japan, Ann. N.Y. Acad. Sci., 141 (1967) 560 568. 15 Mayer, M. and Avi-Dor, Y., Interaction of solvents with membranal and soluble potassium ion-dependent enzymes, Biochem. J., t 16 (1970) 4%54. 16 Percy, E.C. and Carson, J.D., The use of DMSO in tennis elbow and rotator cuff tendonitis: a double-blind study, Med. Sci. Sports Exercise, 13 (1981) 215 219. 17 Reid, K.H., Evans, M.S. and Sharp Jr., J.B., Analgesic-like effects of dimethylsulfoxide on C fiber activity in the cat: evidence for more than one mechanism of action, Soc. Neurosci. Abstr., 8 (1982) 854. 18 Sawada, M. and Sato, M., The effect of dimethylsulfoxide on the neuronal excitability and cholinergic transmission in Aplysia ganglion cells, Ann. N.Y. Acad. Sci., 243 (1975) 337-357. 19 Shealey, C.N., The physiological substrate of pain, Headache, 6 (1966) 101 108.
145
Elsevier Scientific Publishers Ireland Ltd.
NSL 09283
Dimethylsulfoxide (DMSO) blocks conduction in peripheral nerve C fibers: a possible mechanism of analgesia M. Steven Evans a, K e n n e t h H. R e i d b a n d James B. Sharp Jr. c aDivision of Neurology, Department of Internal Medicine, Southern Illinois University School of Medicine, Springfield, IL (USA,), bDepartment of Anatomy and Neurobiology and CDepartment of Veterinary Medicine, University of Louisville School of Medicine, Louisville, K Y (USA) (Received 29 June 1992; Revised version received 3 November 1992; Accepted 5 November 1992)
Key words: Dimethylsulfoxide; Analgesia; Pain; C fiber; Nerve block Dimethylsulfoxide (DMSO) is readily absorbed through skin, and relieves musculoskeletal pain when applied topically to painful areas. We studied the effects of DMSO on C-type nerve fibers, which mediate pain sensation. DMSO was applied directly to exposed cat sural nerves. C fiber conduction velocity was slowed by DMSO, even in low concentrations (5-7% v/v). Higher concentrations completely blocked C fiber conduction, with a minimum blocking concentration of 9%. Onset of nerve block was almost immediate with 15% DMSO or higher concentrations. C fiber blockade may account for analgesia with DMSO.
Dimethylsulfoxide (DMSO) is a solvent with local analgesic properties. It penetrates skin quickly [11], so for analgesia it is applied directly to skin over the painful area. Initial reports on DMSO emphasized its usefulness as a local analgesic [10], and it became a popular remedy for relief from the musculoskeletal pain of arthritis, sprains and strains [13], even though never approved by the Food and Drug Administration for this use [7]. Clinical studies of DMSO found it to be analgesic [2, 4, 12, 14], although some studies found no benefit [16]. Somatic pain is mediated by unmyelinated C type nerve fibers terminating in the skin, muscle and joint capsules [5]. We have studied a possible mechanism of DMSO-induced analgesia by observing the effects of DMSO on conduction in C fibers of cat cutaneous nerves. (Some of these results were presented previously in abstract form [17].) Ninety percent DMSO in water (Eqigisic, Burlington Bio-Medical Co.) was diluted to the desired concentration (expressed as v/v) with deionized water and Hank's Balanced Salt Solution concentrate (Grand Island Biological Company). The final concentrations were (in mM) NaC1 136.9, glucose 11.0, N a H C O 3 15.1, KCI 5.4, CaCI2 1.8, MgSOa.7H20 0.8, KH2PO 4 0.7 and NaH2PO 42H20 0.6. The solute concentration was 280 mM, but total osmolarity was greater due to the DMSO (1588 Correspondence: M.S. Evans, Box 19230, Division of Neurology, Southern Illinois University School of Medicine, Springfield, IL 62794, USA. Fax: (1) (217) 788-5567.
mosmol for a 10% DMSO solution). If necessary, the solution was titrated to pH 7.4 with small amounts of NaH2CO3. Sural nerves of twenty-five random source 2-4 kg cats were studied. Cats were anesthetized with 40 mg/kg sodium pentobarbital intravenously, supplemented as needed to maintain a level of anesthesia sufficient to eliminate withdrawal reflexes. The left sural nerve and its epineurial sheath were carefully microdissected from the underlying muscle and fascia, but left in situ. Proximally, the sural nerve remained connected to the sciatic nerve, and distally remained covered by fascia overlying the gastrocnemius muscle. The nerve's blood supply was left intact. The nerve was placed in a small bath, a slotted glass cylinder with an internal length of 2.2 cm. Bathing medium was injected into the nerve bath manually with a syringe, and removed at a rate of 0.08 ml/min with an infusion pump. The volume of the nerve bath was 0.4 ml, so that a complete change of solution took 5 minutes. A bipolar stimulating electrode was placed on the nerve proximal to the bath, and a bipolar recording electrode distal to it (antidromic stimulation). The cat limb, electrodes and bath were then enclosed in a plastic tent kept at 100% humidity, 36°C. Stimulating and recording electrodes were fabricated from chlorided silver wires. Constant-voltage capacitance-isolated square wave stimuli, 0.5 ms duration, were given at 0.3-3 Hz. Stimulus intensity was set to twice the voltage at C fiber threshold, which ranged from 5 to 10 V
146
A
B
C
2O
10
0
'i
'
I
i
IN 6
~
60
80
°I .m--o, i 20
40
i
i
100
120
, w ~ w i i :
140
i
160
i
i
180 2 0 0
Min~
Fig. 1. A: sural nerve responses are stable. Top: C fiber waveform at beginning of experiment. A fiber responses at the beginning of the record are distorted because of the high gain (which saturates the signal averager) and slow timebase needed to demonstrate C responses. Positiveis upward in all traces. Middle: response 9 h later, with no DMSO treatment. Bottom: same nerve, C fiber response blocked 10 min after application of 10% DMSO. B: DMSO transiently increases spontaneous firing. Top: control sural nerve C fiber response. Middle: 1 min after 5% DMSO, background noise is dramatically increased by spontaneous action potential firing. Bottom: after 20 min in 5% DMSO, spontaneous firing is not apparent. C: DMSO blockade is reversible. A nerve was exposed to DMSO during the times indicated by dotted lines. Control responses (0-20 min) varied in amplitude. 30% and 40% DMSO quickly abolished C fiber responses, with the blockades reversed by washing. In this example, 50% DMSO caused irreversible blockade. (mean of 7.5 V + 0.37 S.E.M.). C fiber responses were identified by their slower conduction velocity, smaller amplitude and higher stimulus threshold compared to A fibers. Conventional electronic devices were used for recording and displaying responses. A general-purpose microcomputer averaged 16 responses, which were then plotted with a chart recorder. The gain and timebase needed to display C responses were incompatible with simultaneously recording A fiber responses. C fiber compound action potential amplitude (most positive point minus most negative point) and latency (to most positive point) were measured. In agreement with previous investigators [5, 6], we found that conduction velocity was stable, but C fiber response amplitudes and shapes varied from minute to minute. Because of this, we used complete block of C fiber responses as an endpoint (Fig. 1A), since this never occurred normally. Complete block was defined as loss of any organized trace of the original waveform. The sural nerve preparation was stable in long-term experiments. Two nerves were followed without drug treatment for nine hours, with no systematic changes in response amplitude or latency (Fig. 1A). During experiments, the health of the nerve was assessed by stroking hairs on the foot, causing action potentials to fire. In no case was responsiveness to this natural stimulus diminished, even after complete blockade of nerve conduction through the bath. Experiments were performed by increasing the D M S O concentration until C fibers were blocked, then lowering the concentration until the block reversed. Immediately after D M S O application, a 1-5 min transient period of increased noise was always seen with concentrations of
5% or more (Fig. 1B). This was due to asynchronous spontaneous firing of axons, transiently reducing the amplitude of evoked C fiber responses. D M S O effects on C fiber response amplitudes were concentration-dependent. Five percent and 7% D M S O tended to reduce C fiber response amplitudes, but the minimum blocking concentration was 9% (Figs. 1A, 2B, and C). In 14 experiments using 9% or 10% D M S O , C fiber responses were invariably reduced, and were completely blocked in 9 experiments (Figs. 1A and 2A). In these 9 experiments, blockade occurred 22.0 min _+ 2.0 after D M S O application. D M S O concentrations of 15% or more reliably blocked C fibers. All nerves exposed to 15% or more D M S O were quickly blocked (mean 6.3 minutes _+ 0.3, n = 15 experiments) (Fig. 2B). Five percent D M S O relieved block caused by higher concentrations (Fig. 2A), but reversal of established blockade was never seen with 9% D M S O or higher concentrations. In 11 experiments (8 nerves), C fiber blockade was produced by 9% to 20% D M S O , followed by application of 5%; recovery was good in l0 of the l l experiments. Because blockade was usually reversible, two or three experiments were performed on some nerves that recovered to 75% of their maximal p r e - D M S O response amplitude. Six nerves were exposed to low concentrations of D M S O for several hours (not illustrated). With such prolonged exposure, 1% to 7% D M S O blocked four nerves 6.25-8 h after application of drug, suggesting that long exposure to low concentrations of D M S O can reduce C fiber transmission. In three of the four nerves, blockade reversed after extended washing.
147
A
B "rime w~ Response
C
Amplimcle
Conce,;U~;on re, Amplitude
120
14 6
.~100 ,o
R
r
t
~
~¢0 60
10% DMS0
:.B ¢= 4o
lS
~ ,o
20
Oo
15% DMSO
2:
qT~,.~~ . . . . . . 1"0 " 20
30
40
50
60
5
70
10
3 15
Minutes
D
E
Time vs. Latency
F
Concentncion vs. latency 4
220
ii,.o[
1BO
160 14(1
RO~
120
7%
-'
100
7% []VI~
0
,'o 2'0 ;
,'o
MintCes
,oo[ go 7o
0'
5'
Con(:entz'e~n [% v/v]
1'o
Fig. 2. DMSO reduces C fiber response amplitude and conduction velocity. A, top: response in 5% DMSO. A, middle: 10% DMSO blocked C fiber responses. A, bottom: two minutes after reapplication of 5% DMSO, response returns. B: time-response curves for DMSO are shown. Changes are expressed as percentage of the mean amplitude of the 3-5 measurments immediately preceding DMSO application. Seven percent and lower concentrations did not block over 3-4 h. The minimum blocking concentration of DMSO was approximately 9%. Most nerves were blocked by a 55 min application of 9% DMSO, and drug application was not continued past that time. Concentrations of 15% and more blocked C responses quickly. Bars indicate standard error of the mean. For clarity, bars are not shown for 0% and 9% DMSO at times less than 35 min. For other observations, if bars are not shown, standard error of the mean is less than symbol size. Small numerals indicate number of experiments. C: Concentration-response curve f6r DMSO. Responses measured 35 min after drug application. Five percent and 7% DMSO slightly reduced response amplitudes, but the difference between these and the control values was not statistically significant (two-tailed t-test). Nine percent DMSO reduced C responses by 50% after 35 min, and eventually blocked most nerves. The difference between 9% DMSO and control is significant (P < 0.05). D: 7% DMSO increased C fiber response latency by 43% over control in this nerve (control latency 78 ms, 7% DMSO 112 ms). E: response latency did not change with time. F: increase in latency is related to DMSO concentration, but not to duration of DMSO exposure. Five percent to 10% DMSO all caused significant increases in response latency over control (P < 0.01). There was no difference in the latencies at 5 min compared to 35 min of DMSO exposure. D M S O r e d u c e d C fiber c o n d u c t i o n velocity (Fig. 2 D F). C o n c e n t r a t i o n s as low as 2% D M S O p r o d u c e d definite increases in r e s p o n s e latency. U n l i k e the effect on r e s p o n s e a m p l i t u d e , w h i c h t o o k tens o f m i n u t e s to dev e l o p with 9% D M S O , the effect o n r e s p o n s e l a t e n c y was a l m o s t i n s t a n t a n e o u s . This effect was stable d u r i n g the time o f d r u g exposure, a n d reversed after d r u g r e m o v a l . R e s p o n s e l a t e n c y i n c r e a s e d b y a l m o s t 200% with 10% D M S O , i m p l y i n g t h a t overall c o n d u c t i o n velocity in the nerve s e g m e n t b e t w e e n s t i m u l a t i n g a n d r e c o r d i n g elect r o d e s was r e d u c e d b y 50%. H o w e v e r , this represents a c o n s e r v a t i v e e s t i m a t e o f the effects o f D M S O , since the nerve s e g m e n t studied, u s u a l l y 5 c m long, was e x p o s e d to D M S O o n l y within the 2.2 c m l o n g nerve b a t h . T h e m e c h a n i s m o f the c o n d u c t i o n b l o c k i n d u c e d b y
D M S O m a y involve p o t a s s i u m c h a n n e l b l o c k a d e . S a w a d a a n d S a t o [18] f o u n d t h a t D M S O c a u s e d a r a p i d m e m b r a n e d e p o l a r i z a t i o n a n d decrease in m e m b r a n e c o n d u c t a n c e o f Aplysia n e u r o n s , c o n s i s t e n t with b l o c k a d e o f leak p o t a s s i u m channels. D M S O s l o w e d a c t i o n potential repolarization, and voltage clamp study s h o w e d t h a t D M S O i n h i b i t e d the d e l a y e d rectifier p o t a s sium channel. T h e i r s t u d y implies t h a t D M S O can p r o duce a d e p o l a r i z i n g nerve b l o c k t h r o u g h i n h i b i t i o n o f b o t h voltage-sensitive a n d voltage-insensitive p o t a s s i u m channels. D e p o l a r i z i n g b l o c k is consistent with results o f the p r e s e n t experiments, in which the t r a n s i e n t increase in electrical noise seen i m m e d i a t e l y after a p p l i c a t i o n o f D M S O suggests t h a t fibers have d e p o l a r i z e d sufficiently to allow s p o n t a n e o u s a c t i o n p o t e n t i a l firing. H o w
148 D M S O m i g h t specifically affect p o t a s s i u m channels is not clear, b u t M a y e r a n d A v i - D o r [15] have suggested that D M S O can c h a n g e the state o f h y d r a t i o n o f the p o tassium ion, which m i g h t alter its ability to p e n e t r a t e ion channels. The m e c h a n i s m o f D M S O ' s ability to p r o f o u n d l y reduce C fiber c o n d u c t i o n velocity is unclear. This effect is fast, stable a n d reversible. It is d e t e c t a b l e with low conc e n t r a t i o n s o f the drug. C o n d u c t i o n velocity r e d u c t i o n m a y also be caused by effects on p o t a s s i u m channels. One possibility is that persistent m e m b r a n e d e p o l a r i z a tion c o u l d facilitate s o d i u m channel i n a c t i v a t i o n , with c o n s e q u e n t slowing o f a c t i o n p o t e n t i a l generation. A seco n d possibility is t h a t an increase in m e m b r a n e resistance due to b l o c k a d e o f p o t a s s i u m channels m i g h t affect cond u c t i o n velocity, b u t in this case the m a g n i t u d e a n d direction o f the effect are n o t easily p r e d i c t e d [9]. C o n d u c tion velocity c o u l d also be r e d u c e d by increasing extracellular electrical resistance [8], b u t u n d e r n o r m a l conditions, c o n d u c t i o n velocity changes a p p r e c i a b l y only with m a n y f o l d increases in extracellular resistance. T h e ability o f 9% D M S O to b l o c k C fibers c o n t r a s t s with previous studies on m y e l i n a t e d A type nerve fibers, in which m u c h larger c o n c e n t r a t i o n s were needed. D a v i s et al. [3] f o u n d t h a t 50% D M S O was necessary to b l o c k muscle twitch in a frog sciatic n e r v e - g a s t r o c n e m i u s muscle p r e p a r a t i o n . Becker [1] f o u n d that 75% D M S O b l o c k e d b o t h A a n d C fibers. Shealey [19] studied a p r o longed small fiber a f t e r d i s c h a r g e in spinal cord, m e d u l l a a n d t e g m e n t u m following s t i m u l a t i o n o f the superficial radial a n d sural nerves, a n d f o u n d it to be b l o c k e d by e x p o s i n g the p e r i p h e r a l nerve to 5-10% D M S O , a result that c o u l d be d u e to b l o c k a d e o f C fibers. T h e present e x p e r i m e n t s suggest t h a t D M S O can be an effective topical analgesic. I f analgesia with D M S O d e p e n d s on b l o c k a d e o f C fibers, one r e q u i r e m e n t is t h a t the d r u g c o n c e n t r a t i o n a r o u n d C fibers leading f r o m the painful a r e a be b r o u g h t to 10% o r m o r e for several minutes. It is likely t h a t this s i t u a t i o n occurs clinically, since D M S O p e n e t r a t e s skin easily, a n d topical a p p l i c a t i o n o f 70 90% is c o m m o n l y used. I n s o f a r as D M S O selectively affects s o m a t i c C fibers, a selective a n a l g e s i a a n d loss o f t e m p e r a t u r e sensation, r a t h e r t h a n c o m p l e t e anesthesia, m a y be expected. L i g h t l y - m y e l i n a t e d A - d e l t a fibers t h a t m e d i a t e s h a r p (first) p a i n were n o t investigated in this study, but if unaffected b y D M S O , w o u l d allow s h a r p p a i n to persist, a n d deep, aching p a i n to be abolished. Deep, aching p a i n is p r e s e n t in arthritis a n d m u s c u loskeletal injuries, the c o n d i t i o n s for which D M S O has often been used.
This w o r k is p a r t o f a thesis s u b m i t t e d by M.S.E. to the G r a d u a t e School o f the U n i v e r s i t y o f Louisville, A u gust, 1984. T h e a u t h o r s t h a n k Jennifer E v a n s a n d D e a n N a r i t o k u for critical r e a d i n g o f the m a n u s c r i p t . 1 Becker, D.P., Young, H.F., Nulsen, F.E. and Jane, J.A., Physiological effects of dimethylsulfoxide on peripheral nerves: possible role in pain relief, Exp. Neurol., 24 (1969) 272-276. 2 Brown, J.H., DMSO its efficacy in acute musculoskeletal problems as evaluated by a 'double blind' study, Industrial Medicine and Surgery, 35 (1967) 777-781. 3 Davis, H.L., Davis, N.L. and Clemons, A.L., Procoagulant and nerve-blocking effects of DMSO, Ann. N.Y. Acad. Sci., 141 (1967) 310 325. 4 Demos, C.H., Beckloff, G.L., Donin, M.N. and Oliver, EM., Dimethylsulfoxide in musculoskeletal disorders, Ann. N.Y. Acad. Sci., 141 (1967)517 523. 5 Douglas, W.W. and Ritchie, J.M., Mammalian non-myelinated nerve fibers, Physiol. Rev., 42 (1962) 297-334. 6 Gasser, H.S., Unmedullated fibers originating in dorsal root ganglia, J. Gen. Physiol., 33 (1950) 650-690. 7 Harter, J.G., The status of dimethylsulfoxide from the perspective of the food and drug administration, Ann. N.Y. Acad. Sci., 411 (1983) 1-5. 8 Hodgkin, A i . , The relation between conduction velocity and the electrical resistance outside a nerve fiber, J. Physiol., 94 (1939) 560570. 9 Hunter, EJ., McNaughton, RA. and Noble, D., Analytical models of propagation in excitable cells, Prog. Biophys., 30 (1975) 99-144. 10 Jacob, S.W., Bischel, M. and Herschler, R.J., Dimethylsulfoxide (DMSO): a new concept in pharmacotherapy, Current Ther. Res., 6 (1964) 134-135. 11 Kolb, K.H., Jaenicke, G., Kramer, M. and Schulze, RE., Absorption, distribution and elimination of labeled dimethylsulfoxide in man and animals, Ann. N.Y. Acad. Sci., 141 (1967) 85-95. 12 Lockie, L.M. and Norcross, B.M., A clinical study on the effects of dimethylsulfoxide in 103 patients with acute and chronic musculoskeletal injuries and inflammations, Ann. N.Y. Acad. Sci., 141 (1967) 599-602. 13 MacGrady, R, The Persecuted Drug: The Story of DMSO, Doubleday and Company, Garden City, New York, 1973, 335 pp. 14 Matsumoto. J., Clinical trials of dimethylsulfoxide in rheumatoid arthritis patients in Japan, Ann. N.Y. Acad. Sci., 141 (1967) 560 568. 15 Mayer, M. and Avi-Dor, Y., Interaction of solvents with membranal and soluble potassium ion-dependent enzymes, Biochem. J., t 16 (1970) 4%54. 16 Percy, E.C. and Carson, J.D., The use of DMSO in tennis elbow and rotator cuff tendonitis: a double-blind study, Med. Sci. Sports Exercise, 13 (1981) 215 219. 17 Reid, K.H., Evans, M.S. and Sharp Jr., J.B., Analgesic-like effects of dimethylsulfoxide on C fiber activity in the cat: evidence for more than one mechanism of action, Soc. Neurosci. Abstr., 8 (1982) 854. 18 Sawada, M. and Sato, M., The effect of dimethylsulfoxide on the neuronal excitability and cholinergic transmission in Aplysia ganglion cells, Ann. N.Y. Acad. Sci., 243 (1975) 337-357. 19 Shealey, C.N., The physiological substrate of pain, Headache, 6 (1966) 101 108.