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Electroencephalography and Clinical Neurophysiology, 1 9 7 9 , 4 6 : 3 5 1 - - 3 5 4 © E l s e v i e r / N o r t h - H o l l a n d Scientific Publishers, Ltd. Laboratory
note
NERVE CONDUCTION NEUROTOXICITY 1
VELOCITY
AND
REFRACTORY
PERIOD
AS PARAMETERS
OF
D.V. R O B E R T S a n d I.E. T R O L L O P E
Department of Physiology, University of Liverpool, P.O. Box 14 7, Liverpool (England) ( A c c e p t e d for p u b l i c a t i o n : O c t o b e r 27, 1 9 7 8 )
Nerve c o n d u c t i o n v e l o c i t y a n d r e f r a c t o r y p e r i o d are t w o p a r a m e t e r s w h i c h have b e e n used e x t e n s i v e l y to p r o v i d e e v i d e n c e o f f u n c t i o n a l a b n o r m a l i t y in t h e n e r v o u s s y s t e m , p r e f e r a b l y in a d v a n c e o f o v e r t clinical signs a n d s y m p t o m s or h i s t o p a t h o l o g i c a l changes. T h e value of these f u n c t i o n a l p a r a m e t e r s as i n d i c a t o r s of small c h a n g e s f r o m t h e n o r m a l is l i m i t e d b y a n y variability o f m e a s u r e d values. In a d d i t i o n t o i n n a t e biological variability, o t h e r f a c t o r s such as t e m p e r a t u r e , age a n d n u t r i t i o n a l s t a t e are k n o w n t o a f f e c t c o n d u c t i o n v e l o c i t y a n d r e f r a c t o r y period. T h e purpose of this p a p e r is t o describe a s y s t e m o f measurem e n t of these p a r a m e t e r s in vitro in t h e r a t a n d t o q u a n t i f y t h e individual d e v i a t i o n f r o m g r o u p m e a n values.
Method and material T h e rats used in these e x p e r i m e n t s were a l b i n o males, s t r a i n CFHB. T h e i r ages r a n g e d f r o m 12 t o 13 m o n t h s at w h i c h t i m e age-related c h a n g e s are negligible ( M i y o s h i a n d G o t o 1 9 7 3 ) . T h e tissues for e x a m i n a t i o n were r e m o v e d u n d e r p e n t o b a r b i t o n e s o d i u m a n a e s t h e s i a . I n t h e case o f t h e $1--$3 r o o t s , t h e tissues e x p o s e d b y l a m i n e c t o m y were k e p t in situ u n d e r o x y g e n a t e d m a m m a l i a n saline s o l u t i o n (LUey 1 9 5 6 ) and the roots removed one by one and transferred to t h e m e a s u r i n g c h a m b e r . In this way t h e vascular supply a n d o x y g e n a t i o n o f t h e r e m a i n i n g tissues a w a i t i n g e x a m i n a t i o n were preserved. For measurement of conduction velocity and r e f r a c t o r y p e r i o d , t h e nerves were p l a c e d o n a n elect r o d e a s s e m b l y c o n s i s t i n g of 17 silver wires ( d i a m e t e r = 0 . 0 4 6 r a m ) s p a c e d at 2.5 m m intervals. D i r e c t plug-in c o n n e c t i o n s were m a d e t o t h e s e e l e c t r o d e s for the purposes of stimulation, recording and earthing t h e p r e p a r a t i o n . T h e w h o l e e l e c t r o d e a s s e m b l y was
1 T h i s w o r k was s u p p o r t e d financially b y Shell I n t e r n a t i o n a l e R e s e a r c h Mij. B.V., T h e Hague, T h e Netherlands.
e n c l o s e d in t h e m e a s u r i n g c h a m b e r w h i c h was vent i l a t e d w i t h a m i x t u r e o f 5% CO2 a n d 95% 0 2 , satur a t e d w i t h w a t e r v a p o u r , t h e t e m p e r a t u r e b e i n g maint a i n e d a t t h e r e q u i r e d value -+0.5°C b y a s u r r o u n d i n g w a t e r jacket.
Electrophysiological technique
(1) Conduction velocity measurements Single s u p r a m a x i m a l electrical stimuli ( 2 0 p s e c d u r a t i o n ) were applied t o o n e e n d o f t h e n e r v e t r u n k a n d t h e r e s u l t a n t c o m p o u n d a c t i o n p o t e n t i a l s were r e c o r d e d at 4 p o i n t s a l o n g t h e nerve. A f t e r p h o t o g r a p h y of t h e C R O display, a s t r a i g h t line was d r a w n t h r o u g h t h e fast rising p h a s e of each p o t e n t i a l a n d p r o j e c t e d t o i n t e r s e c t t h e baseline. T h e t e m p o r a l sepa r a t i o n s o f t h e 4 i n t e r s e c t i o n s were m e a s u r e d a n d calc u l a t e d , using a 0.1 msec t i m e - m a r k e r o n t h e s e c o n d b e a m o f t h e CRO. T h e linear regression o f c o n d u c t i o n t i m e o n c o n d u c t i o n d i s t a n c e was c a l c u l a t e d b y t h e m e t h o d o f least s q u a r e s a n d used t o p r o v i d e a value for t h e m a x i m u m c o n d u c t i o n velocity (MCV). In a typical e x p e r i m e n t , s o l u t i o n of t h e e q u a t i o n y = a0 + al • x gave a value for al o f 50.7 m / s e c (MCV) w i t h a c o r r e l a t i o n c o e f f i c i e n t , r 2, o f 0 . 9 9 9 2 . T h e value o f t h e o t h e r c o e f f i c i e n t , a0, was 0.66 m m i n d i c a t i n g t h a t , o w i n g t o l o n g i t u d i n a l s p r e a d o f s t i m u l a t i n g curr e n t , t h e site o f origin of t h e a c t i o n p o t e n t i a l was n o t e x a c t l y at t h e c a t h o d e b u t at a p o i n t 0.66 m m n e a r e r t o t h e r e c o r d i n g e l e c t r o d e s . C o n d u c t i o n velocity b a s e d o n t h e t i m e o f arrival at t h e first r e c o r d i n g elect r o d e a n d t h e d i s t a n c e of this e l e c t r o d e f r o m t h e c a t h o d e w o u l d be o v e r e s t i m a t e d b y a b o u t 10%, b u t t h e use of t h e linear regression m e t h o d avoids this error.
(2) Refractory period measurements Pairs of s u p r a m a x i m a l electrical s t i m u l i ( 2 0 psec d u r a t i o n ) were applied t o t h e e n d o f t h e n e r v e t r u n k , t h e t i m e interval b e t w e e n t h e first a n d s e c o n d s t i m u l i o f e a c h pair was varied over t h e range 0 . 6 - - 4 . 0 msec using a D i g i t i m e r pulse g e n e r a t o r (Devices, L t d . ) a n d
352
D.V. R O B E R T S , I.E. T R O L L O P E
the r e s u l t a n t c o m p o u n d a c t i o n p o t e n t i a l s were displayed o n a s t o r a g e - t y p e C R O and p h o t o g r a p h e d . The a m p l i t u d e o f the s e c o n d a c t i o n p o t e n t i a l s were measured and s u b t r a c t e d f r o m the m a x i m u m a m p l i t u d e a t t a i n e d by the s e c o n d action p o t e n t i a l . This difference or ' e x t i n c t i o n value', e x p r e s s e d as a p e r c e n t a g e o f t h e m a x i m u m value, is a measure o f the p r o p o r t i o n o f nerve fibres w h i c h failed to r e s p o n d t o the s e c o n d stimulus, i.e. were r e f r a c t o r y . T h e r e was f o u n d t o be an inverse linear relation b e t w e e n t h e l o g a r i t h m o f the e x t i n c t i o n value and t h e c o r r e s p o n d i n g t i m e interval b e t w e e n the first and seco n d s t i m u l u s and this may be e x p r e s s e d in the f o r m y = a • e bx, w h e r e y is the e x t i n c t i o n value, x is t h e t i m e interval b e t w e e n stimuli and a and b are coefficients. F o r each nerve p r e p a r a t i o n the c o e f f i c i e n t s a and b were f o u n d b y s o l u t i o n o f the e x p o n e n t i a l e q u a t i o n for the 5 s h o r t e s t s t i m u l u s intervals, i.e. over t h e s t e e p part o f the e x t i n c t i o n value vs. stimulus interval curve, and were used to calculate the. t i m e interval c o r r e s p o n d i n g to 100% e x t i n c t i o n w h e n all fibres in the p r e p a r a t i o n were r e f r a c t o r y . This interval is r e f e r r e d t o as the ' m i n i m u m r e f r a c t o r y p e r i o d ' (MRP).
Results (1) Experiments to control the effect o f interelectrode distance on conduction velocity measurements and to demonstrate the stability o f the preparation while in the measuring cham ber The c o n d u c t i o n velocities o f 13 dorsal and 11 ventral r o o t s were m e a s u r e d with t h e r e c o r d i n g elect r o d e s 2.5, 5.0 and again 2.5 m m apart, so t h a t the length o f nerve over w h i c h the m e a s u r e m e n t was m a d e was e i t h e r 1 or 2 cm. The m e a n velocity in all t h e r o o t s t e s t e d in this way was n o t significantly altered by t h e change in r e c o r d i n g e l e c t r o d e separat i o n (paired t-test). In all s u b s e q u e n t m e a s u r e m e n t s , h o w e v e r , the 2.5 m m s e p a r a t i o n was used. The t i m e t a k e n to carry o u t t h e 3 m e a s u r e m e n t s o f c o n d u c t i o n velocity in each r o o t was 10 min and t h e r e was n o significant change over this p e r i o d (paired t-test). On the o t h e r h a n d , slowing o f cond u c t i o n velocity due to anoxia was o b s e r v e d w i t h i n 1 rain o f s t o p p i n g t h e gas f l o w t h r o u g h t h e m e a s u r i n g c h a m b e r and so it was c o n c l u d e d t h a t t h e nerve prepa r a t i o n w o u l d r e m a i n in a c o n s t a n t and viable state f o r t h e 10 min r e q u i r e d t o m e a s u r e c o n d u c t i o n velocity a n d r e f r a c t o r y p e r i o d for each nerve p r e p a r a t i o n .
(2) Comparison o f MCV and MRP in ventral and dorsal roots, and in saphenous nerves When t h e m a x i m u m c o n d u c t i o n velocities o f nerve preparations t a k e n f r o m 6 rats were m e a s u r e d at a temperature
of
32°C
significant d i f f e r e n c e s ( P =
0.001) were f o u n d to exist b e t w e e n ventral and dorsal r o o t and s a p h e n o u s nerve values (Table I). Using t h e same nerve p r e p a r a t i o n s , significant diff e r e n c e s (P= 0.001) were also f o u n d in the m e a n m i n i m u m r e f r a c t o r y p e r i o d s (Table I). While the results in Table I w o u l d a p p e a r to suggest an association b e t w e e n m a x i m u m c o n d u c t i o n velocity and mini m u m r e f r a c t o r y p e r i o d , n o c o r r e l a t i o n was f o u n d b e t w e e n these p a r a m e t e r s w i t h i n each o f the 3 nerve groups e x a m i n e d .
(3) Temperature coefficients o f maximum conduction velocity and minimum refractory period While the t e m p e r a t u r e o f the measuring c h a m b e r was m a i n t a i n e d within 0.5°C o f any s t a t e d t e m p e r a ture it was c o n s i d e r e d necessary to find o u t t h e e x t e n t to w h i c h the 2 p a r a m e t e r s c o u l d vary w i t h i n this small t e m p e r a t u r e range. A c c o r d i n g l y , m a x i m u m c o n d u c t i o n velocity and m i n i m u m r e f r a c t o r y p e r i o d were m e a s u r e d in all 3 nerve p r e p a r a t i o n s at t e m p e r a tures o f 20, 24, 28, 32 and 36°C (Table II). A statistically significant (P = 0.001) linear relation was f o u n d b e t w e e n t e m p e r a t u r e and m a x i m u m c o n d u c t i o n velocity with regression c o e f f i c i e n t s o f 3 . 9 + - 0 . 1 3 (S.E.M.), 3.25_+ 0.12 and 2.4-+ 0.17 m / s e c / ° C for ventral and dorsal r o o t s and s a p h e n o u s nerves respectively. The d i f f e r e n c e s b e t w e e n these regression c o e f f i c i e n t s are significant ( P = 0 . 0 0 1 ) . Because these values are small in c o m p a r i s o n with t h e 95% c o n f i d e n c e limits o f m a x i m u m c o n d u c t i o n velocities m e a s u r e d at a n o m i n a l 32°C, it is c o n c l u d e d t h a t any e r r o r arising f r o m t e m p e r a t u r e changes within t h e range 3 1 . 5 - - 3 2 . 5 ° C is unlikely to invalidate the c o n c l u s i o n s based on c o n d u c t i o n velocity measurements. In the case o f the m i n i m u m r e f r a c t o r y p e r i o d , the changes p r o d u c e d by t e m p e r a t u r e were m o r e complex (Table II) and were best d e s c r i b e d by an inverse p o w e r law relationship, i.e. m i n i m u m r e f r a c t o r y period was f o u n d to be inversely related t o s o m e p o w e r o f the t e m p e r a t u r e . The p o w e r values for t h e 3 nerve
TABLE I C o m p a r i s o n o f m a x i m u m c o n d u c t i o n v e l o c i t y (MCV)
and m i n i m u m r e f r a c t o r y period (MRP) in ventral a n d dorsal r o o t s and s a p h e n o u s nerves. T e m p e r a t u r e = 32°C. Root/ nerve
MCV ( m e a n + 1 S.E.M., m/sec)
Ventral 81.45 + 0.96, n = 24 Dorsal 73.96 + 1.24, n = 26 S a p h e n o u s 4 9 . 1 2 + 1.25, n = 6
MRP ( m e a n + 1 S.E.M., m/sec) 0.63 + 0.01, n = 27 0.52 +- 0.01, n = 28 0.47 _+ 0.01, n = 6
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T A B L E II M C V a n d M R P values over t h e t e m p e r a t u r e r a n g e 2 0 - - 3 6 ° C ( m e a n + 1 S.E.M.). Temperature
MCV (m/sec)
M R P (msec)
(°c) 20 24 28 32 36
Ventral
Dorsal
Saphenous
Ventral
Dorsal
Saphenous
3 2 . 7 0 + 0.89 n=ll 55.14 + 2.17 n=ll 7 1 . 3 0 + 1.12 n =11 8 1 . 4 5 + 0.96 n = 24 9 9 . 4 7 +_ 1.81 n=12
3 6 . 3 7 + 1.89 n=12 49.80 + 0.92 n=12 6 2 . 8 7 _+ 1.51 n=ll 7 3 . 9 6 + 1.24 n = 26 9 9 . 0 0 +_ 1.71 n=12
26.75 n=4 33.13 n=3 39,58 n=3 49.12 n = 6 62.79 n=3
2.61 + 0.12 n=6 1.40 + 0.04 n=ll 0.81 + 0.02 n =12 0.63 + 0.01 n = 27 0.43 +_ 0.01 n = 12
2.04 + 0.06 n=12 1.06 + 0.05 n=12 0.67 _+ 0.03 n=10 0.52 _+ 0.01 n=28 0.39 +_ 0.01 n=12
1.27 n=l 0.91 n=3 0.48 n=3 0.47 n=6 0.34 n=4
p r e p a r a t i o n s were n o t significantly d i f f e r e n t f r o m each o t h e r a n d h a d a m e a n value of 2.69. T h i s corres p o n d s to a change, at 32°C, o f 0.05 m s e c / ° C w h i c h is small c o m p a r e d w i t h t h e 95% c o n f i d e n c e l i m i t s o f t h e m i n i m u m r e f r a c t o r y periods for all 3 n e r v e prepa r a t i o n s , m e a s u r e d at 32°C. E r r o r s in r e f r a c t o r y period values due to t e m p e r a t u r e f l u c t u a t i o n s w i t h i n t h e range 3 1 . 5 - - 3 2 . 5 ° C m a y , t h e r e f o r e , b e discounted.
Discussion T h e results o f this s t u d y s h o w t h a t t h e r e are sign i f i c a n t d i f f e r e n c e s in t h e values for m a x i m u m cond u c t i o n v e l o c i t y a n d m i n i m u m r e f r a c t o r y p e r i o d in v e n t r a l a n d dorsal spinal r o o t s a n d s a p h e n o u s nerves, a n d t h a t t h e s e d i f f e r e n c e s exist over t h e t e m p e r a t u r e range 2 0 - - 3 6 ° C . N o s y s t e m a t i c e v a l u a t i o n o f t h e s e p a r a m e t e r s has b e e n carried o u t previously, b u t Rasm i n s k y a n d Sears ( 1 9 7 2 ) q u o t e d a r a n g e o f values for c o n d u c t i o n v e l o c i t y , m e a s u r e d in i s o l a t e d l u m b a r ventral r o o t s , o f 3 8 . 0 7 - - 7 3 . 6 0 m / s e c at 37°C. T h e s e values are smaller t h a n t h o s e n o w r e p o r t e d b u t this m a y be d u e t o t h e d i f f e r e n t t e c h n i q u e s a n d r o o t s used in t h e t w o studies. S i m a a n d J a n k o w s k a ( 1 9 7 5 ) f o u n d MCV values o f 39.6 +- 2.0 m / s e c ( m e a n + S.E.M.) for l u m b a r dorsal r o o t s , m e a s u r e d at 2 1 - - 2 2 ° C , w h i c h agree closely w i t h t h e c o r r e s p o n d i n g values in T a b l e II. Values p r e v i o u s r e p o r t e d f o r t h e s a p h e n o u s n e r v e of t h e r a t were 52.9 +- 15.2 m / s e c ( m e a n -+ S.D.) for MCV a n d 1.65 ± 0.52 m ] s e c {mean-+ S.D.) for t h e a b s o l u t e r e f r a c t o r y period, m e a s u r e d a t 3 7 - - 3 8 ° C ( T r u h a u t et al. 1 9 7 3 ) . B o t h values d i f f e r widely f r o m t h o s e n o w r e p o r t e d , p a r t i c u l a r l y in t h e i r c o e f f i c i e n t s
+ 1.28 + 0.95 _+ 2.84 + 1.25 +_ 4.03
+ 0.13 _+ 0.02 + 0.01 + 0.01
of variation, w h i c h are five t i m e s as large, b u t again this m a y be due to d i f f e r e n c e s in t e c h n i q u e .
Summary The maximum conduction velocity (MCV) and m i n i m u m r e f r a c t o r y p e r i o d ( M R P ) were m e a s u r e d in r a t sacral ventral a n d dorsal r o o t s , a n d s a p h e n o u s nerves. A t 32°C values for M C V were 8 1 . 4 5 , 7 3 . 9 6 a n d 4 9 . 1 2 m / s e c for t h e v e n t r a l a n d dorsal r o o t s , a n d s a p h e n o u s nerves respectively, w i t h c o r r e s p o n d i n g t e m p e r a t u r e c o e f f i c i e n t s o f 3.9, 3.25 a n d 2.14 m ] s e c / °C. Values of M R P at 32°C for t h e 3 n e r v e preparat i o n s were 0.63, 0.52 a n d 0.47 msec, w i t h a n average t e m p e r a t u r e c o e f f i c i e n t o f 0.04 m s e c / ° C at 32°C.
R~sum~ Vitesse de conduction et pdriode refractaire du nerf c o m m e paramdtres de neurotoxicitd La vitesse de c o n d u c t i o n m a x i m a l e ( M C V ) et la p~riode r e f r a c t a i r e m i n i m a l e ( M R P ) du n e r f s a p h ~ n e et des racines dorsales e t v e n t r a l e s c h e z le r a t o n t ~t~ mesur~es in vitro. A 32°C, n o u s a v o n s o b t e n u respect i v e m e n t les valeurs s u i v a n t e s de M C V p o u r les racines v e n t r a l e s et dorsales et le n e r f s a p h ~ n e : 8 1 , 4 5 , 7 3 , 9 6 , e t 4 9 , 1 2 m / s e c ; les c o e f f i c i e n t s de t e m p d r a t u r e corres p o n d a n t s ~ t a i e n t : 3,9, 3,25 et 2,4 m / s e c / ° C . Les valeurs de la M R P q u e n o u s a v o n s o b t e n u e s p o u r ces m ~ m e s n e r f s fi 32°C ~ t a i e n t : 0,63, 0,52 et 0,47 m s e c , avec u n c o e f f i c i e n t m o y e n de t e m p 4 r a t u r e de 0 , 0 4 m s e c ] ° C ~ 32°C.
354 References
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