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Colchicine prevents recovery of nerve conduction at chronic demyelination
Brain Research, 519 (1990) 50-56
50
Elsevier BRES 15547
Colchicine prevents recovery of nerve conduction at chronic demyelination Sigal Liverant and Hamutal Meiri Department of Physiology and Biophysics, Faculty of Medicine and The Rappaport Family Institute for Research in the Medical Sciences, Technion-lsrael Institute of Technology, 31096 Haifa (Israel) (Accepted 21 November 1989)
Key words: Lysophosphatidylcholine; Sodium channel; Fast axonal transport; Action potential; Antibody to sodium channel
A colchicine cuff was applied to rat sciatic nerve proximal to a demyelinating region produced by a focal injection of lysophosphatidylcholine (LPC). The colchicine cuff prevented the recovery of function normally seen within 6-8 days after LPC-induced demyelination. Colchicine blocked the delivery of sodium channels to the demyelinated region and induced their accumulation proximal to the cuff. The dual effect of colchicine in blocking both the recovery of impulse propagation through the demyelinated region and the delivery of sodium channels suggests a central role for fast axonal transport of sodium channels in the recovery of function at demyelination. INTRODUCTION D e m y e l i n a t i o n is p r o d u c e d in n u m e r o u s p a t h o p h y s i o logical c o n d i t i o n s 35'4°'49. I n acute d e m y e l i n a t i o n , n e r v e c o n d u c t i o n t h r o u g h the d e m y e l i n a t e d r e g i o n is completely b l o c k e d 5'6'3°'37'49. H o w e v e r , with chronic d e m y e l i n a t i o n , a n d o f t e n b e f o r e r e m y e l i n a t i o n is achieved, n e r v e c o n d u c t i o n t h r o u g h the d e m y e l i n a t e d r e g i o n is r e s t o r e d 5-7'37'48'49'51'52. Several physiological, m o r p h o logical, a n d b i o c h e m i c a l studies have i n d i c a t e d s o d i u m c h a n n e l r e c r u i t m e n t b y the d e m y e l i n a t e d axon a n d e m p h a s i z e d the i m p o r t a n c e o f this process for the r e c o v e r y of i m p u l s e p r o p a g a t i o n t h r o u g h the d e m y e l i n a ted a r e a 5-7'37'48'49. T h e pools c o n t r i b u t i n g s o d i u m channels to d e m y e l i n a t e d axons have n o t b e e n fully identified. H o w e v e r , a role for S c h w a n n cells 9'11'23'47, lateral m o bility f r o m a d j a c e n t n o d e s 2'4°, a n d c h a n n e l delivery from t h e cell b o d y ( w h e r e they are synthesized s) to the d e m y e l i n a t e d region, by m e a n s of a x o n a l t r a n s p o r t 12'3t, have b e e n suggested. I n the p r e s e n t study, the i m p o r t a n c e of fast a x o n a l t r a n s p o r t in s u p p o r t i n g b o t h the r e c o v e r y of c o n d u c t i o n a n d the supply of s o d i u m channels to the d e m y e l i n a t e d r e g i o n was tested. Fast a x o n a l t r a n s p o r t was arrested by the classical fast t r a n s p o r t b l o c k e r colchicine 16'21'22"27.
subjected to double treatment, i.e. by injection and operation, as follows: (1) demyelination was induced focally, as previously described37, by injection of 20 #1 of 0.025% lysophosphatidyleholine (LPC) from egg yolk (Sigma, St. Louis, MO) in Ringer solution, 30-35 mm from the spinal cord, using a Hamilton syringe inserted under the epineurium (upper panel, Fig. 1). (2) A cuff was made from a 5 × 10 mm piece of oxidized cellulose (Ethicon W-1915), which was soaked in 5 mM of colchicine (Sigma) in Ringer solution and then delicately applied around the sciatic nerve, 10-15 mm from the spinal cord (upper panel, Fig. 1). The cuff remained in place for 2-3 days and was then absorbed into the body fluid. At the end of the operation, the muscles and skin were sutured back into place. The animals were revived, and each was kept in a separate cage. The colehicine concentration employed (5 mM) was selected according to observations made by Devor and Govrin-Lipmann12, who used this approach to arrest increased excitability in lesioned peripheral axons. A larger concentration (50 mM) was toxic (>70% of the animals died), and a smaller concentration (0.5 mM) produced none of the effects described below. The double-treated colchicine-LPC (C-L) group was compared to 3 control groups: (1) a Ringer-Ringer (R-R) group, which was used as a control for the operation; (2) a colchicine-Ringer (C-R) group, which was used as a control for the cuff alone; and (3) a Ringer-LPC (R-L) group, which was used as a control for the demyelination alone. The left nerves were left untreated.
Behavioral studies Rats were tested daily. A rat was placed on the floor of a silent room (3 x 4 m) and allowed to walk. When walking difficulties developed, the rats avoided using all or a few toes of the treated hind leg when tested. In a second test, rats were lifted from the floor, and the number of toes of the hind leg open upon lifting was counted. Scores are given between 0 (no use of toes for walking and no opening of toes upon being lifted from floor) and 5 (all toes open and used for walking).
MATERIALS AND METHODS
Electrophysiological studies Demyelination and cuff application Forty Charles River rats (200-300 g b. wt.) were anesthetized with ether, and their right sciatic nerves were exposed. Each nerve was
Two to 10 rats in each group were anesthetized by 50 mg/kg b. wt. of pentobarbitone, and their right and left sciatic nerves were separately isolated, desheathed, and mounted on a recording
Correspondence: H. Meiri, 2600 Netherland Avenue, Apt. 717, Riverdale, Bronx, NY 10463, U.S.A. 0006-8993/90/$03.50 ~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
51
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Fig. 1. Measurements of compound action potential. Upper panel: schematic drawing of electrophysiological experiments in relation to cuff and injection regions. 1-4 represent the recording and stimulating electrodes, each suitable for both functions: 1-2, measurements and recording in the segment proximal to the cuff; 2-3, measurements and recording across the cuff; 3-4, measurements and recording across the injection region; 1-4, measurements from the entire nerve. A - D : measurements from the different nerve segments (as above). A: R-R nerves 8 days postoperatively. B,C: R-L nerves 2 days (B) and 8 days (C) postoperatively. Arrowhead in C denotes electrodes 3-4 at the point of the ectopic action potential. The double records I and II in C (electrodes 2-3) denote different recording sensitivity (I, as indicated by the calibration bar; II half sensitivity). D: C-L nerves 8 days postoperatively. Note the conduction time difference between C and D electrodes. For further details, see Table III. chamber. Stimulation was given at 1 Hz. All measurements were made at room temperature in a Ringer solution composed of (in mM): NaCI, 154; KCL, 5.6; CaCI2, 1.8; MgCI2, 0.9; and 10 mM Tris, pH 7.4, adjusted with 1 N HCI. To analyze conduction changes in each segment of the treated nerves in each group, 4 electrodes were placed on the nerves (upper panel, Fig. 1), as follows: (1) stimulation and recording of conduction proximal to the cuff was made between electrodes 1 and 2. (2) Stimulation and recording with electrodes 2 and 3 enabled evaluation of conduction through the cuff. (3) Electrodes 3 and 4 showed impulse characteristics while crossing the demyelinated region. (4) Measurements between electrodes 1 and 4 enabled the evaluation of all segmental effects. Similar recordings were made along the left, untreated nerves, but segmental differences there were insignificant (not shown). Thus, the parameters for all segments of the left nerves were pooled for each experimental group.
Histological studies Left and right nerves of 3-5 rats in each group were removed,
fixed in 3% paraformaldehyde, and washed in phosphate-buffered saline (PBS), pH 7.4. Bundles were isolated, mounted, and examined by light microscopy at 63 x 10 magnification. In each nerve, 100 large (>15 pm diameter) axons 1-3 mm long were examined at each segment, and the number of axons with any specific lesion was counted. Axon diameter was measured, and the mean axon diameter was calculated. For segmental demyelination, axons were scored if the bare axolemma was longer than 20 #m. For paranodal demyelination, axons were scored if the nodal region was above 5 gm. Degenerating axons had a dark cytoplasm with many organelles inside. Demyelination and degeneration values are the means + S.D. of the frequency of each lesion in all nerves in a given group. A total of 300-500 axons were examined per group.
Binding studies Two sodium channel-specific antibodies were used: a monoclonal antibody (mAb), denoted SC-72-383'34'35, and a polyclonal antibody, anti-C1 ÷ (ref. 36). mAb SC-72-38 was generated against sodium channel-enriched membrane fractions and was found to modify sodium channel function by changing the voltage dependency of both channel activation and inactivation3. Anti-C1 + was generated against a synthetic peptide corresponding to a part of the $4 segment of internal repeat 1 of the sodium channel 3s. This antibody was previously found to modify sodium channel inactivation 36. Both antibodies were employed in radioimmunoassays to detect the number of antigenic determinants associated with sodium channel function along all segments of the sciatic nerves. mAb SC-72-38. Sciatic nerves of anesthetized rats were isolated, desheathed, and placed on a roller, where the injection and cuff regions were marked. Nerves were cut into 10 5-mm segments, and corresponding segments of 5-10 rats were pooled, homogenized in 5 ml PBS, and centrifuged at 1000 g for 10 min. The supernatant was removed and centrifuged at 40,000 g for 45 min. The pellet was resuspended in 0.5 ml PBS, and samples of 20-30 #l were adsorbed into a 2.5 x 2.5 cm filter (Schlicher and Schull). After drying, the filters were blocked for 1 h with 1% bovine serum albumin and 0.05% Tween-20 in PBS, followed by overnight incubation with the mAb (50 #g/ml) from ascitic fluid used after ultrafiltration with Amicon minidialyser membranes (XM-035, cutoff 35 kDa). The bound antibody was detected with lzsI-labeled goat anti-mouse IgG. Washing with PBS and PBS-Tween was performed between steps. For control, we used normal mouse IgG (Zymed). Anti-Ct ÷. Segments (5 mm each) proximal to the cuff, the cuff segments, the injection segments, and the segments distal to the injection were pooled from all nerves, homogenized, and processed as described above for mAb SC-72-38. The bound antibody was detected by 125I-protein A. For controls, we used normal rabbit IgG or anti-Cl ÷ preblocked with Cl +. RESULTS
Behavioral studies I m m e d i a t e l y after t h e animals h a d r e c o v e r e d f r o m t h e a n e s t h e s i a , t h e toes o f t h e right h i n d leg w e r e p a r a l y z e d in all o p e r a t e d a n i m a l s , w h o a v o i d e d s t e p p i n g o n this leg while walking. L a t e r , r e c o v e r y o f f u n c t i o n was slowly a c h i e v e d (Table I). T w o days a f t e r t h e o p e r a t i o n , and to a g r e a t e r e x t e n t at 8 days, t h e ability o f t h e a n i m a l s in the R - R g r o u p to o p e n t h e t o e s o f t h e i r right leg u p o n b e i n g lifted f r o m t h e f l o o r was r e s t o r e d in s o m e o r all o f t h e toes (Table I). O n t h e 8th day ( b u t n o t o n t h e 2nd day), a similar d e g r e e o f r e c o v e r y was s e e n in t h e R - L g r o u p (Table I). T h e r e c o v e r y o f f u n c t i o n was s o m e w h a t s l o w e r in the C - R g r o u p and was c o m p l e t e l y a b s e n t in t h e C - L g r o u p (Table I). I n t e r e s t i n g l y , no effect was f o u n d in the
52 TABLE I Behavioral recovery
Recovery was measured using the parameter of mean number of hind-leg toes open upon lifting rat from floor. Values are means + S.D. and were compared using a two-tailed t-test. *, P < 0.02; **, P < 0.002, versus R-R group in each fight leg. Open toes (n)
Time
R-R
R-L
C-R
C-L
Left hind leg Left hind leg Right hind leg Right hind leg Right hind leg
30 min 8 days 30 min 2 days 8 days
5 5 0 1.1+0.8 3.6+0.5
5 5 0 0.2+0.05" 3.4+0.5
5 5 0 1.2+0.7 2.5+0.5
5 5 0 0"* 0"*
untreated left leg, indicating the local and non-systemic effects of the operation and of the application of the colchicine cuff and the LPC injection. This relatively superficial and subjective analysis is useful in many ways: (1) It helps in setting the timetable for comparing the experimental groups. (2) It shows that comparison should be made between the R - R group and the other groups, and not between the left and right sides of each animal. (3) It indicates that the colchicine cuff prevents the recovery of function from a focal injection of LPC, and that the effect is specific and greater than that induced by the colchicine cuff alone (C-R group). Histological examination
To evaluate the type and level of injury with each treatment, segments of right and left nerves were
TABLE II Histological lesions
Histological analysis was performed as described under Materials and Methods. Values (means + S.D.) are the frequency of the lesion (%) in a total of 300-500 axons, except for axon diameter (pm). They were compared using two-tailed unpaired t-test. *, P < 0.01; **, P < 0.005; ***, P < 0.001, versus R-R group in each fight nerve. Parameter
R-R
R-L
Left whole nerve Segmental demyelination 2 + 1 2 +2 Paranodal demyelination 8 + 5 9 +7 Axonal degeneration Axonal diameter ~m) 20 + 7 21 +3 -
C-R
3+1 8+7 -
24+9
C-L
2+2 7+4 2+2 23+9
analyzed histologically by light microscopy (Table II). As expected 5'24'37, the effectiveness of LPC in producing segmental demyelination in rat sciatic nerve was very high and was found in approx. 60% of all axons in both the C-L and R - L groups, but not in the C-R and R - R groups (Table II). The focal effect of L P C 24'37 w a s demonstrated by the lower frequency of segmental demyelination at the cuff region versus the injection region of the C-L and C-R groups (Table II). Paranodal demyelination also developed as a result of both the cuff operation and the injection into the right nerves of the R - R group and in all examined right (but not left) nerves (Table II), indicating that this phenomenon could be attributed primarily to the damage associated with the operation. However, in the injection region of the C-L and R-L groups, most axons had paranodal demyelination, and this lesion was less frequent in the C-R group. In contrast, in the cuff region, paranodal demyelination was most frequent in the C-R group, and there were fewer with paranodal demyelination in all the other groups. Degenerating axons were seen in both the cuff region and the injection region of the C-R and C-L groups (Table II). The degenerating axons were found mostly at the external perimeters of the nerve trunk, i.e. closer to the colchicine cuff. Thus, the degeneration could be attributed to the direct harmful effect of colchicine on survival of the axons. Degeneration was not found in the core axons, consistent with the finding of Jackson and Diamond 27. Degenerating profiles were rarely found in the R-L and R - R groups, indicating that the degeneration was not due to the cuff itself. Usually, a degenerating axon had paranodal demyelination, as previously demonstrated in many other cases 24'5°. An increase in axon diameter was found in the C-R and C-L groups at the cuff but not the injection region (Table II). The increased diameters were consistent with anticipated swelling of the axons at the regions where axonal transport is arrested 16'22'27. The implications for nerve conduction of all these histological changes will be discussed below. The results shown in Table II emphasize the different local effects at the cuff and injection regions. The left nerves were virtually unaffected. Electrophysiological measurements
Right nerve injection region Segmentaldemyelination 2+ 2 Paranodal demyelination 27 + 5 Axonal degeneration 5+ 4 Axonal diameter (pm) 22 + 5
58 +8"** 67 +9"** 9 +3 27 +9
15+8"* 30+7 30+5** 33+6
60+7"** 71+9"** 42+8*** 32+8
Right nerve cuff region Segmental demyelination 5 + 2 Paranodal demyelination 13 + 3 Axonal degeneration 3+ 4 Axonal diameter (pm) 24 + 3
4+ 3 25 + 7 7+ 4 23 _+7
12+6 35+3** 40 + 8 37+9
15_+7 30+4** 45 + 9*** 35+7
The influence of all treatments on nerve conduction was tested in parallel. Nerves removed within 1 h postoperatively did not conduct action potential (not shown). Two days postoperatively, recovery of conduction was seen in the R - R group. Eight days postoperatively, conduction was found in the R-R, R-L, and C-R groups. There was almost no recovery in the C-L group. The results, summarized in Table III, showed that the
53 TABLE III Electrophysiological parameters
Action potential amplitude (APA), action potential duration at 90% amplitude (APD9o), and conduction velocity (CV) were recorded at supramaximal stimulation. Current threshold at infinite stimulus (lr) was recorded from strength-duration curves. All measurements were made 8 days after cuff and injection were applied. Measurements are described for the cuff and injection regions (right nerve) and for the whole nerve (left nerve). Values are means + S.D. and were compared using a two-tailed unpaired t-test. *, P < 0.02; **, P < 0.002, versus R-R group in each right nerve. Parameter
R-R
R- L
C-R
C- L
APA (mV) Left whole nerve Right nerve cuff region Right nerve injection region
4.5 + 3.9 2.2 + 0.8 2.1 + 1.6
6.0 + 0.4 7.2 + 1.6" 4.4 + 1.8
4.0 + 3.5 0.6 + 0.2 1.5 + 1.3
5.2 + 0.6 1.1 + 0.4 0.4 + 0.3**
APDgo (ms) Left whole nerve Right nerve cuffregion Right nerve injection region
1.4 + 0.3 1.1 + 1.1 0.5 ___0.2
1.1 + 0.4 0.8 + 0.3 3.7 + 0.4**
0.9 + 0.3 0.6 + 0.3 0.6 __+0.4
0.5 + 0.3 0.6 + 0.1 0.7 + 0.3
c v (m/s) Left whole nerve Right nerve cuffregion Right nerve injection region
50.5 + 24.7 43.6 + 21.8 49.1 + 3.1
56.7 + 15.8 29.1 + 5.9**
45.0 + 4.5 53.8 + 15.0 50.0 + 5.0
37.5 + 17.7 31.1 + 16.7 37.5 + 17.7
I~ (gA) Left whole nerve Right nerve cuffregion Right nerve injection region
150 _+77 92 _+30 139 + 42
187 _+25 251 _+ 180 245 _+7**
161 + 155 160 _+40
185 _+78 156 + 114 194 _+37
p a r a m e t e r s of the left nerves in all groups were similar, regardless of the t r e a t m e n t , reinforcing our earlier impression from the behavioral and histological studies that t h e r e was no significant systemic effect of either L P C or colchicine on the m e a s u r e d parameters. T h e e x a m p l e s given in Fig. 1 A - D and the results s u m m a r i z e d in Table I I I show that with sham t r e a t m e n t ( R - R group), nerve conduction was partially i m p a i r e d , as indicated by reduction of the action potential a m p l i t u d e in the right versus left nerves of the R - R group (Table III). The degree o f injury induced by the surgical m a n i p u l a t i o n was increased from proximal to distal (Fig. 1A, Table III). In view of all the preceding, in each s e g m e n t all p a r a m e t e r s in each t r e a t e d group were c o m p a r e d to those of the corresponding segment of the right nerves o f the R - R group and not to those of the left nerves of the t r e a t e d group. T h e impact of L P C d e m y e l i n a t i o n is shown in Fig. 1B,C and Table III. Two days after L P C injection, there was practically no conduction through the LPC injection s e g m e n t (electrodes 3 - 4 , Fig. 1B). Eight days after the o p e r a t i o n , nerve conduction was r e s t o r e d (Fig. 1C). P r o p a g a t i o n through the d e m y e l i n a t e d region had a significantly low conduction velocity, and the threshold current (It) was significantly higher. These results are u n d e r s t a n d a b l e if one assumes that the conducting axons either are n o n - m y e l i n a t e d or have short-segmented internodes 19'24'a5'44. In either case, nerve conduction is antic i p a t e d to be slower 18'26'52. A l s o consistent with this
assumption was the finding of an action potential with two peaks, a larger and a smaller one (electrodes 3 - 4 , Fig. 1C). The first c o r r e s p o n d e d to the impulse g e n e r a t e d close to the recording e l e c t r o d e across the d e m y e l i n a t e d region, while the second, smaller one could be an ectopic one g e n e r a t e d at the d e m y e l i n a t e d segment, which, due to i m p e d a n c e mismatch 29'52, could be p r o p a g a t e d electrotonically. Such records have been p r e d i c t e d in simulation analysis of nerve conduction at d e m y e l i n a t i o n as described using various m a t h e m a t i c a l m o d e l s 28'29'41"52. The presence of a colchicine cuff proximal to the L P C - i n d u c e d focal d e m y e l i n a t i o n (C-L group) p r e v e n t e d the recovery of both action potential a m p l i t u d e and duration at the L P C - i n j e c t e d region (Fig. 1D, Table III). The tiny action potential d e t e c t e d in these circumstances had a normal conduction velocity, suggesting that the conducting axons were the few that were not injured by L P C and therefore c o n d u c t e d the typical saltatory and rapid action potentials 18'26. Recording at the injection region showed that the colchicine cuff was harmful in itself (C-R group, Table III), as demonstrated by the reduction in action potential amplitude and duration in the C - R versus R - R groups. However, conduction velocity and threshold currents in the C-R group were similar to those in the R - R group. These results suggest that, although colchicine induced degeneration of a substantial number of axons, the injured axons degenerated and simply disappeared from the records, while the remaining ones were more or less normal.
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Fig. 2. Binding of mAb SC-72-38 to sciatic nerves. Pooled values of left nerves are shown for each group (see text). Arrows indicate cuff (left) and injection (right) regions. Lower panel: schematic diagram of surgical lesion with respect to the different segments. It is interesting to compare the changes in action potential parameters between the injection and cuff regions. In the latter, all action potential parameters of the R-L group were larger than in the R-R group, except for nerve conduction velocity, which was significantly slower. These results can be understood using the prediction made by the mathematical models of impulse propagation from myelinated to demyelinated segments 4' 1 3 , 1 5 , 2 8 , 2 9 , 4 1 , 5 2 . These models predict that at the segments preceding the demyelinated segments, the propagating action potential would have a slower conduction, since the cable space constant is influenced by the increased capacity of the membrane in the demyelinated segments ahead 28'29'52. Also, if the demyelinated region recruits more sodium channels, the impulse at the preceding segments would be expected to be larger 4'15'29'41"52. The increased duration should be anticipated, considering that the rate of rise of the action potential decreases as
TABLE IV Binding of anti-C 1÷ to sciatic nerve
Th binding of sodium channel-specific polyclonal antibodies generated against a synthetic peptide denoted C1÷ (anti-C1÷) was measured. Values were normalized to protein content determined by the Lowry procedure32. When more than one experiment was performed, values are the means + S.D. of 5 experiments. Nervesegment
R-R
R-L
C-R
C-L
Left whole nerve 262+240 250+31 300+180 250+171 Right nerve Proximalto cuff 73 + 41 200 1430 + 60 52,397 + 532 Cuffregion 202 + 53 261 + 14 26 + 11 46 Injection region 347 + 47 163 + 15 163 + 39 70 Distal to injection 98 + 30 162 + 36 28 + 13 75 + 27
a result of the capacitance increase at the demyelinated region15,28,29, 41" The increased duration and amplitude and the decreased conduction velocity at the cuff region were not seen in the C-L group. The differences between the R-L and C-L groups were consistent with the failures in impulse propagation through the demyelinated segment in the C-L group (Table III). The widening of the axons at the cuff region in the C-L group adds to the decreased safety factor for impulse propagation from the preceding into the demyelinated region 4'20'29,39,41. The C-R group was less injured than the C-L group, indicating that the combination of axonal transport block and demyelination prevented recovery from the focal demyelination. B i n d i n g studies
Involvement of sodium channel delivery from cell bodies to the LPC-injected region was demonstrated by measuring the number and distribution of sodium channels along the treated right and non-treated left nerves in all experimental groups. It was discovered that 8 days after injection of LPC, the number of binding sites to both mAb SC-72-38 (Fig. 2) and anti-C1 ÷ (Table (IV) at the LPC-injected segment of the R-L group was 2- to 4-fold larger than in the nerves of the R-R group or in the left, untreated nerves. These results confirm previous studies performed in our laboratory 35'37. The placement of a colchicine cuff between the cell bodies and the LPC-injected segment (C-L group) prevented the recruitment of sodium channel antigenic determinants at the demyelinated region (Fig. 2, Table IV). Accumulation of antibody-binding sites was found proximal to the colchicine cuff (C-R group) (Fig. 2, Table IV). In the C-L group, accumulation proximal to the colchicine cuff was greater than in the C-R group (Fig. 2, Table IV). Altogether, results of the binding experiments showed that at LPC demyelination of rat sciatic nerve, there was a focal increase in antigenic determinants associated with functional domains of the sodium channel. Channel recruitment could be prevented by the classical fast axonal transport blocker colchicine 12'21'27. The latter was equally efficient in blocking delivery of sodium channels to the rest of the axon, even without LPC-induced demyelination, confirming the central role of fast axonal transport in carrying sodium channels in uninjured nerves, as previously reported 31. From the results obtained in the C-L group, it appears that at demyelination more channels were being transported that way, as indicated by the larger accumulation in the C-L versus C-R groups. As the colchicine cuff prevented both the supply of sodium channel antigenic determinants and the recovery of function at demyelination, the two processes
55 are probably interrelated. Thus, our results can serve to emphasize the central role of sodium channel delivery by axoplasmic transport in functional recovery from demyelination. DISCUSSION An increase in sodium channels at demyelination of mammalian nerves has been implied in many studies 5' 19,35,37,40,42,48,51. A somewhat different observation was made in frog sciatic nerve, in which LPC was found to be capable of inducing focal demyelination 46. However, when the loose patch-clamp technique was applied to recording sodium currents at the demyelinated axons, the detected current density in nodes corresponded to 10002000 sites//~m2 (refs. 43, 46), and at the demyelinated axons the current density corresponded to 60-100 channels//~m2. In the demyelinated region, the sodium channel density immediately after demyelination and during the next 6 days did not change 46. In parallel, impulse propagation through the demyelinated region was not restored during the first 6 days 46. The differences between frog and rat in regard to recovery from demyelination can be attributed simply to the differences in species and in the measuring techniques. However, they can also be attributed to the difference in body temperature between frog (4-20 °C) and rat (37 °C). It is well known that fast axonal transport is slowed by cooling from 37 to 20 °C and is completely arrested at 4 °C x3'22. Thus, the lack of increase in sodium channels and the failure in recovery of function at demyelinated axons of the frog 8 days postoperatively may be a result of the lower efficiency of fast axonal transport in delivering sodium channels in frogs versus rats. A comparative study should be performed to examine this assumption. There is one conceptual concern with our findings with regard to specificity of coichicine action, especially on a long-term scale. To study the long-term fate of the REFERENCES 1 Allen, R.D., Weiss, D.G., Hayden, J.H., Brown, D.T. and Fugiwake, H., Gliding movement and bidirectional transport along single native microtubules from squid axoplasm: evidence for an active role of microtubules in cytoplasm transport, J. Cell Biol., 100 (1985) 1736-1752. 2 Angelides, K.J., Elmer, L.W., Loftus, D. and Elson, E., Distribution and lateral mobility of voltage-dependent sodium channels in neurons, J. Cell Biol., 106 (1988) 1911-1925. 3 Barhanin, J., Meiri, H., Romey, G., Pauron, D. and Lazdunski, M., A monoclonal immunotoxin acting on the Na ÷ channel, with properties similar to those of a scorpion toxin, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 1842-1846. 4 Berkinblit, M.B., Vvedenkaya, N.D., Guedenko, L.S., Kovalev, S.A., Kholopov, A.V., Fomin, S.V. and Chailakhyan, L.M., Computer investigation of the features of conduction of a nerve impulse along fibres with different degrees of widening, Bio-
LPC-treated axons after colchicine treatment, a similar study has been performed in which the animals were examined after 1-3 months. The results are now being summarized (Meiri and Liverant, in preparation). However, it is already possible to say that the main finding is not degeneration of the demyelinated axons distal to the site of the LPC injection. Rather, we found remyelination and a large degree of sprouting. A full description of the long-term effects is now being prepared for publication. The present study emphasizes the central role of fast axonal transport in the recovery of impulse propagation through a demyelinating axon. However, we are not suggesting that this is the only mechanism responsible for recovery of function at demyelination. Substantial evidence can be cited to support the mechanism of channel supply from the Schwann cells 9-11'33'44'47. Our results show merely that the ability of the other mechanisms to contribute to functional recovery at demyelination is dependent on the well-being of fast axonal transport. Using more detailed analysis of the components of fast axonal transport 22 associated with sodium channel transport and employing electron microscopy with its larger resolution power 25, it may be possible to understand in detail how sodium channels are carried to the demyelinated segments. Using the approach of video-enhanced microscopy 1'45 and employing channel-specific markers, we intend to follow the cascade of events involved in sodium channel delivery to the demyelinated region. Concomitant analysis of changes in the potassium channels 1°'47'51 is required to complete the picture.
Acknowledgements. We are most grateful to Dr. M. Sammar for preparing and purifying anti-C1÷, and to O. Amir, S. Kopler, D. Dicker, and U. Metzer for helping to establish the surgical procedure, behavioral testing, and electrophysiological measurements. This study was supported by a basic research grant (No. 480.87) of the Israel Academy of Sciences and Humanities. physics, 15 (1970) 1121-1131. 5 Bostock, H. and Sears, T., The internodal axon membrane: electrical excitability and continuous conduction in segmental demyelination, J. Physiol. (Lond.), 280 (1978) 273-301. 6 Brismar, T., Potential clamp analysis of membrane currents in rat myelinated nerve fibres, J. Physiol. (Lond.), 298 (1980) 171-180. 7 Brismar, T., Specific permeability properties of demyelinated rat nerve fibres, Acta Physiol. Scand., 113 (1981) 167-176. 8 Caterail, W.A., Structure and function of voltage-sensitive ion channels, Science, 242 (1988) 50-61. 9 Chiu, S.Y., Changes in excitable membrane properties in Schwann cells of adult rabbit sciatic nerves following nerve transection, J. Physiol. (Lond.), 396 (1988) 173-188. 10 Chiu, S.Y., Ritchie, J.M., Rogart, R.B. and Stagg, D.A., A quantitative description of membrane currents in rabbit myelinated nerve, J. Physiol. (Lond.), 292 (1979) 149-166. 11 Chiu, S.Y., Shrager, P. and Ritchie, J.M., Neuronal-type Na ÷
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and K + channels in rabbit cultured Schwann cells, Nature (Lond.), 311 (1984) 156-157. Devor, M. and Govrin-Lipmann, R., Axoplasmic block reduces ectopic impulse generation in injured peripheral nerves, Pain, 16 (1983) 73-79. Dodge, EA.J. and Cooley, T.W., Action potential of the motoneuron, IBM J. Res. Div., 17 (1973) 219-229. Droz, B. and Leblond, EC., Migration of proteins along the axons of the sciatic nerve, Science, 137 (1962) 1047-1048. Dun, ET., The length and diameter of the node of Ranvier, IEEE Trans. Biomed. Eng., 1 (1970) 21-24. Dustin, P., Microtubules, Springer-Verlag, Berlin, 1978, pp. 167-180. EUisman, M.H., Wiley, C.A., Lindsey, J.D. and Wurtz, C.C., Structure and function of the cytoskeleton and endomembrane system at the node of Ranvier. In The Node of Ranvier, Academic Press, New York, 1984, pp. 153-181. FitzHugh, R., Computation of impulses initiation and saltatory conduction in myelinated nerve fibre, Biophys. J., 2 (1962) 11-21.
19 Foster, R.E., Whalen, C.C. and Waxman, S.G., Reorganization of the axon membrane in the demyelinated nerve fibers: morphological evidence, Science, 210 (1980) 661-663. 20 Goldman, L. and Albus, J.S., Computation of impulse conduction in myelinated fibres: theoretical basis of the velocitydiameter relation, Biophys. J., 8 (1968) 596-607. 21 Goodman, C.S., Corey, S. and Heitler, W.J., Electrical properties of insect neurons with spiking and non-spiking somata: normal, axotomized, and colchicine-treated neurons, J. Exp. Biol., 83 (1979) 95-121. 22 Grafstein, B. and Forman, D.S., IntraceUular transport in neurons, Physiol. Rev., 60 (1980) 1167-1283. 23 Gray, P.T.A. and Ritchie, J.M., Ion channels in Schwann and glial cells, Trends Neurosci., 8 (1985) 411-415. 24 Hall, S.M. and Gregson, N.A., The in vivo and ultrastructural effects of injection of lysophosphatidylcholine into myelinated peripheral nerve fibres of the adult mouse, J. Cell Sci., 9 (1971) 769-789. 25 Heimovich, B., BoniUa, E., Casadi, J. and Barchi, R., Immunochemical localization of the voltage-dependent sodium channels using polyclonal antibodies against the purified protein, J. Neurosci., 4 (1984) 2259-2308. 26 Huxley, A.E and Stampfli, R., Evidence for saltatory conduction in peripheral myelinated nerve fibres, J. Physiol. (Lond.), 108 (1949) 315-339. 27 Jackson, P. and Diamond, J., Colchicine block of cholinesterase transport in rabbit sensory nerves without interference with the long viability of the axons, Brain Research, 130 (1977) 579-584. 28 Khodorov, B.I. and Timin, E.N., Nerve impulse propagation along non-uniform fibres, Prog. Biophys. Mol. Biol., 30 (1975) 145-184. 29 Koles, Z.J. and Rasminsky, M., A computer simulation of conduction in demyelinated nerve fibres, J. Physiol. (Lond.), 227 (1972) 1049-1052. 30 Lafontaine, S., Rasminsky, M., Saida, T. and Sumner, A.J., Conduction block in rat myelinated fibres following acute exposure to anti-galactocerebroside serum, J. Physiol. (Lond.), 252 (1982) 287-306. 31 Lombet, A., Laduron, P., Mourre, C., Jocomet, Y. and Lazdunski, M., Axonal transport of the voltage-dependent Na channel protein identified by its tetrodotoxin binding site in rat sciatic nerves, Brain Research, 345 (1985) 153-158. 32 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin-phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 33 Meador-Woodruff, J.H., Yoshino, J.E., Bigbee, J.W., Lewis, B.L. and DeVries, H.G., Differential proliferative responses of
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cultured Schwann cells to axolemma and myelin-enriched fraction. II. Morphological studies, J. Neurocytol., 14 (1985) 619-635. Meiri, H., Detection of cell surface sodium channels by monoclonal antibodies - - could the channels become exposed to the external surface and 'down regulated' by binding to antibodies?, Brain Research, 368 (1986) 188-192. Meiri, H., Baum, Z. and Rosenthal, Y., Dynamic changes in sodium channels at demyelinated axons, Prog. Neurobiol., 32 (1989) 159-179. Meiri, H., Spira, G., Marei, S., Namir, M., Schwartz, A., Komoriya, A., Kosower, E.M. and Palti, Y., Mapping a region associated with Na channel inactivation using antibodies to a synthetic peptide corresponding to a part of the Na channel, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 5058-5062. Meiri, H., Steinberg, R. and Medalion, B., Detection of sodium channel distribution in rat sciatic nerve following lysophosphatidylcholine-induced demyelination, J. Membr. Biol., 92 (1986) 47-56. Noda, M., Ikeda, T., Kayano, T., Suzuki, H., Takashima, H., Kurasaki, M., Kakahashi, H., Nakayama, H. and Numa, S., Existence of distinct sodium channel messenger RNAs in rat brain, Nature (Lond.), 320 (1986) 188-192. Parnas, I., Hochstein, S. and Parnas, H., Theoretical analysis of parameters leading to frequency modulation along an inhomogeneous axon, J. Neurophysiol., 39 (1976) 909-923. Rasminsky, M., Physiology of conduction in demyelinated axons. In S.G. Waxman (Ed.), Physiology and Pathobiology of Axons, Raven Press, New York, 1978, pp. 361-376. Ravenko, S.V., Timin, E.N. and Khodorov, B.I., Propagation of nerve impulse from myelinated part of axon in unmyelinated terminal, Biophysics, 18 (1973) 1074-1078. Ritchie, J.M. and Rang, H.E, Extraneuronal saxitoxin binding sites in rabbit myelinated nerve, Proc. Natl. Acad. Sci. U.S.A., 83 (1983) 2803-2805. Ritchie, J.M. and Rogart, R.B., The density of sodium channels in mammalian myelinated nerve fibers and the nature of the axonal membrane under the myelin sheath, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 211-215. Rosenthal, Y. and Meiri, H., Detection of sodium channels in demyelinated sciatic nerve axons and Schwann cells, Brain Research, submitted. Schnapp, B.J. and Reese, T.S., New development in understanding rapid axonal transport, Trends Neurosci., 9 (1986) 155-162. Shrager, P., The distribution of sodium and potassium channels in single demyelinated axons of the frog, J. Physiol. (Lond.), 392 (1987) 587-602. Shrager, P., Chiu, S.Y. and Ritchie, J.M., Voltage-dependent sodium and potassium channels in mammalian cultured Schwann cells, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 948-952. Smith, K.J., Bostock, H. and Hall, S.M., Saltatory conduction precedes remyelination in axons demyelinated with phosphatidylcholine, J. Neurol. Sci., 54 (1982) 13-31. Smith, K.J. and Hall, S.M., Nerve conduction during peripheral demyelination and remyelination, J. Neurol. Sci., 48 (1980) 201-219. Spencer, P. and Thomas, P.K., Ultrastructural studies of the dying-back process. II. The sequestration and removal by Schwann cells and oligodendrocytes of organelles from normal and diseased axons, J. Neurocytol., 3 (1974) 63-78. Waxman, S.G. and Ritchie, J.M., Organization of ion channels in the myelinated nerve fiber, Science, 228 (1985) 1502-1507. Waxman, S.G. and Wood, S.L., Impulse conduction in inhomogeneous axons: effects of variation in voltage-sensitive ionic conductances on invasion of demyelinated-axon segments and preterminal fibers, Brain Research, 294 (1984) 111-122.
50
Elsevier BRES 15547
Colchicine prevents recovery of nerve conduction at chronic demyelination Sigal Liverant and Hamutal Meiri Department of Physiology and Biophysics, Faculty of Medicine and The Rappaport Family Institute for Research in the Medical Sciences, Technion-lsrael Institute of Technology, 31096 Haifa (Israel) (Accepted 21 November 1989)
Key words: Lysophosphatidylcholine; Sodium channel; Fast axonal transport; Action potential; Antibody to sodium channel
A colchicine cuff was applied to rat sciatic nerve proximal to a demyelinating region produced by a focal injection of lysophosphatidylcholine (LPC). The colchicine cuff prevented the recovery of function normally seen within 6-8 days after LPC-induced demyelination. Colchicine blocked the delivery of sodium channels to the demyelinated region and induced their accumulation proximal to the cuff. The dual effect of colchicine in blocking both the recovery of impulse propagation through the demyelinated region and the delivery of sodium channels suggests a central role for fast axonal transport of sodium channels in the recovery of function at demyelination. INTRODUCTION D e m y e l i n a t i o n is p r o d u c e d in n u m e r o u s p a t h o p h y s i o logical c o n d i t i o n s 35'4°'49. I n acute d e m y e l i n a t i o n , n e r v e c o n d u c t i o n t h r o u g h the d e m y e l i n a t e d r e g i o n is completely b l o c k e d 5'6'3°'37'49. H o w e v e r , with chronic d e m y e l i n a t i o n , a n d o f t e n b e f o r e r e m y e l i n a t i o n is achieved, n e r v e c o n d u c t i o n t h r o u g h the d e m y e l i n a t e d r e g i o n is r e s t o r e d 5-7'37'48'49'51'52. Several physiological, m o r p h o logical, a n d b i o c h e m i c a l studies have i n d i c a t e d s o d i u m c h a n n e l r e c r u i t m e n t b y the d e m y e l i n a t e d axon a n d e m p h a s i z e d the i m p o r t a n c e o f this process for the r e c o v e r y of i m p u l s e p r o p a g a t i o n t h r o u g h the d e m y e l i n a ted a r e a 5-7'37'48'49. T h e pools c o n t r i b u t i n g s o d i u m channels to d e m y e l i n a t e d axons have n o t b e e n fully identified. H o w e v e r , a role for S c h w a n n cells 9'11'23'47, lateral m o bility f r o m a d j a c e n t n o d e s 2'4°, a n d c h a n n e l delivery from t h e cell b o d y ( w h e r e they are synthesized s) to the d e m y e l i n a t e d region, by m e a n s of a x o n a l t r a n s p o r t 12'3t, have b e e n suggested. I n the p r e s e n t study, the i m p o r t a n c e of fast a x o n a l t r a n s p o r t in s u p p o r t i n g b o t h the r e c o v e r y of c o n d u c t i o n a n d the supply of s o d i u m channels to the d e m y e l i n a t e d r e g i o n was tested. Fast a x o n a l t r a n s p o r t was arrested by the classical fast t r a n s p o r t b l o c k e r colchicine 16'21'22"27.
subjected to double treatment, i.e. by injection and operation, as follows: (1) demyelination was induced focally, as previously described37, by injection of 20 #1 of 0.025% lysophosphatidyleholine (LPC) from egg yolk (Sigma, St. Louis, MO) in Ringer solution, 30-35 mm from the spinal cord, using a Hamilton syringe inserted under the epineurium (upper panel, Fig. 1). (2) A cuff was made from a 5 × 10 mm piece of oxidized cellulose (Ethicon W-1915), which was soaked in 5 mM of colchicine (Sigma) in Ringer solution and then delicately applied around the sciatic nerve, 10-15 mm from the spinal cord (upper panel, Fig. 1). The cuff remained in place for 2-3 days and was then absorbed into the body fluid. At the end of the operation, the muscles and skin were sutured back into place. The animals were revived, and each was kept in a separate cage. The colehicine concentration employed (5 mM) was selected according to observations made by Devor and Govrin-Lipmann12, who used this approach to arrest increased excitability in lesioned peripheral axons. A larger concentration (50 mM) was toxic (>70% of the animals died), and a smaller concentration (0.5 mM) produced none of the effects described below. The double-treated colchicine-LPC (C-L) group was compared to 3 control groups: (1) a Ringer-Ringer (R-R) group, which was used as a control for the operation; (2) a colchicine-Ringer (C-R) group, which was used as a control for the cuff alone; and (3) a Ringer-LPC (R-L) group, which was used as a control for the demyelination alone. The left nerves were left untreated.
Behavioral studies Rats were tested daily. A rat was placed on the floor of a silent room (3 x 4 m) and allowed to walk. When walking difficulties developed, the rats avoided using all or a few toes of the treated hind leg when tested. In a second test, rats were lifted from the floor, and the number of toes of the hind leg open upon lifting was counted. Scores are given between 0 (no use of toes for walking and no opening of toes upon being lifted from floor) and 5 (all toes open and used for walking).
MATERIALS AND METHODS
Electrophysiological studies Demyelination and cuff application Forty Charles River rats (200-300 g b. wt.) were anesthetized with ether, and their right sciatic nerves were exposed. Each nerve was
Two to 10 rats in each group were anesthetized by 50 mg/kg b. wt. of pentobarbitone, and their right and left sciatic nerves were separately isolated, desheathed, and mounted on a recording
Correspondence: H. Meiri, 2600 Netherland Avenue, Apt. 717, Riverdale, Bronx, NY 10463, U.S.A. 0006-8993/90/$03.50 ~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
51
cuff
injection
3, j® -
tins
R-LPC~Sh}
(~)
R-LPC~d)
(~
GOL-LPC(Sd)
(~)
Fig. 1. Measurements of compound action potential. Upper panel: schematic drawing of electrophysiological experiments in relation to cuff and injection regions. 1-4 represent the recording and stimulating electrodes, each suitable for both functions: 1-2, measurements and recording in the segment proximal to the cuff; 2-3, measurements and recording across the cuff; 3-4, measurements and recording across the injection region; 1-4, measurements from the entire nerve. A - D : measurements from the different nerve segments (as above). A: R-R nerves 8 days postoperatively. B,C: R-L nerves 2 days (B) and 8 days (C) postoperatively. Arrowhead in C denotes electrodes 3-4 at the point of the ectopic action potential. The double records I and II in C (electrodes 2-3) denote different recording sensitivity (I, as indicated by the calibration bar; II half sensitivity). D: C-L nerves 8 days postoperatively. Note the conduction time difference between C and D electrodes. For further details, see Table III. chamber. Stimulation was given at 1 Hz. All measurements were made at room temperature in a Ringer solution composed of (in mM): NaCI, 154; KCL, 5.6; CaCI2, 1.8; MgCI2, 0.9; and 10 mM Tris, pH 7.4, adjusted with 1 N HCI. To analyze conduction changes in each segment of the treated nerves in each group, 4 electrodes were placed on the nerves (upper panel, Fig. 1), as follows: (1) stimulation and recording of conduction proximal to the cuff was made between electrodes 1 and 2. (2) Stimulation and recording with electrodes 2 and 3 enabled evaluation of conduction through the cuff. (3) Electrodes 3 and 4 showed impulse characteristics while crossing the demyelinated region. (4) Measurements between electrodes 1 and 4 enabled the evaluation of all segmental effects. Similar recordings were made along the left, untreated nerves, but segmental differences there were insignificant (not shown). Thus, the parameters for all segments of the left nerves were pooled for each experimental group.
Histological studies Left and right nerves of 3-5 rats in each group were removed,
fixed in 3% paraformaldehyde, and washed in phosphate-buffered saline (PBS), pH 7.4. Bundles were isolated, mounted, and examined by light microscopy at 63 x 10 magnification. In each nerve, 100 large (>15 pm diameter) axons 1-3 mm long were examined at each segment, and the number of axons with any specific lesion was counted. Axon diameter was measured, and the mean axon diameter was calculated. For segmental demyelination, axons were scored if the bare axolemma was longer than 20 #m. For paranodal demyelination, axons were scored if the nodal region was above 5 gm. Degenerating axons had a dark cytoplasm with many organelles inside. Demyelination and degeneration values are the means + S.D. of the frequency of each lesion in all nerves in a given group. A total of 300-500 axons were examined per group.
Binding studies Two sodium channel-specific antibodies were used: a monoclonal antibody (mAb), denoted SC-72-383'34'35, and a polyclonal antibody, anti-C1 ÷ (ref. 36). mAb SC-72-38 was generated against sodium channel-enriched membrane fractions and was found to modify sodium channel function by changing the voltage dependency of both channel activation and inactivation3. Anti-C1 + was generated against a synthetic peptide corresponding to a part of the $4 segment of internal repeat 1 of the sodium channel 3s. This antibody was previously found to modify sodium channel inactivation 36. Both antibodies were employed in radioimmunoassays to detect the number of antigenic determinants associated with sodium channel function along all segments of the sciatic nerves. mAb SC-72-38. Sciatic nerves of anesthetized rats were isolated, desheathed, and placed on a roller, where the injection and cuff regions were marked. Nerves were cut into 10 5-mm segments, and corresponding segments of 5-10 rats were pooled, homogenized in 5 ml PBS, and centrifuged at 1000 g for 10 min. The supernatant was removed and centrifuged at 40,000 g for 45 min. The pellet was resuspended in 0.5 ml PBS, and samples of 20-30 #l were adsorbed into a 2.5 x 2.5 cm filter (Schlicher and Schull). After drying, the filters were blocked for 1 h with 1% bovine serum albumin and 0.05% Tween-20 in PBS, followed by overnight incubation with the mAb (50 #g/ml) from ascitic fluid used after ultrafiltration with Amicon minidialyser membranes (XM-035, cutoff 35 kDa). The bound antibody was detected with lzsI-labeled goat anti-mouse IgG. Washing with PBS and PBS-Tween was performed between steps. For control, we used normal mouse IgG (Zymed). Anti-Ct ÷. Segments (5 mm each) proximal to the cuff, the cuff segments, the injection segments, and the segments distal to the injection were pooled from all nerves, homogenized, and processed as described above for mAb SC-72-38. The bound antibody was detected by 125I-protein A. For controls, we used normal rabbit IgG or anti-Cl ÷ preblocked with Cl +. RESULTS
Behavioral studies I m m e d i a t e l y after t h e animals h a d r e c o v e r e d f r o m t h e a n e s t h e s i a , t h e toes o f t h e right h i n d leg w e r e p a r a l y z e d in all o p e r a t e d a n i m a l s , w h o a v o i d e d s t e p p i n g o n this leg while walking. L a t e r , r e c o v e r y o f f u n c t i o n was slowly a c h i e v e d (Table I). T w o days a f t e r t h e o p e r a t i o n , and to a g r e a t e r e x t e n t at 8 days, t h e ability o f t h e a n i m a l s in the R - R g r o u p to o p e n t h e t o e s o f t h e i r right leg u p o n b e i n g lifted f r o m t h e f l o o r was r e s t o r e d in s o m e o r all o f t h e toes (Table I). O n t h e 8th day ( b u t n o t o n t h e 2nd day), a similar d e g r e e o f r e c o v e r y was s e e n in t h e R - L g r o u p (Table I). T h e r e c o v e r y o f f u n c t i o n was s o m e w h a t s l o w e r in the C - R g r o u p and was c o m p l e t e l y a b s e n t in t h e C - L g r o u p (Table I). I n t e r e s t i n g l y , no effect was f o u n d in the
52 TABLE I Behavioral recovery
Recovery was measured using the parameter of mean number of hind-leg toes open upon lifting rat from floor. Values are means + S.D. and were compared using a two-tailed t-test. *, P < 0.02; **, P < 0.002, versus R-R group in each fight leg. Open toes (n)
Time
R-R
R-L
C-R
C-L
Left hind leg Left hind leg Right hind leg Right hind leg Right hind leg
30 min 8 days 30 min 2 days 8 days
5 5 0 1.1+0.8 3.6+0.5
5 5 0 0.2+0.05" 3.4+0.5
5 5 0 1.2+0.7 2.5+0.5
5 5 0 0"* 0"*
untreated left leg, indicating the local and non-systemic effects of the operation and of the application of the colchicine cuff and the LPC injection. This relatively superficial and subjective analysis is useful in many ways: (1) It helps in setting the timetable for comparing the experimental groups. (2) It shows that comparison should be made between the R - R group and the other groups, and not between the left and right sides of each animal. (3) It indicates that the colchicine cuff prevents the recovery of function from a focal injection of LPC, and that the effect is specific and greater than that induced by the colchicine cuff alone (C-R group). Histological examination
To evaluate the type and level of injury with each treatment, segments of right and left nerves were
TABLE II Histological lesions
Histological analysis was performed as described under Materials and Methods. Values (means + S.D.) are the frequency of the lesion (%) in a total of 300-500 axons, except for axon diameter (pm). They were compared using two-tailed unpaired t-test. *, P < 0.01; **, P < 0.005; ***, P < 0.001, versus R-R group in each fight nerve. Parameter
R-R
R-L
Left whole nerve Segmental demyelination 2 + 1 2 +2 Paranodal demyelination 8 + 5 9 +7 Axonal degeneration Axonal diameter ~m) 20 + 7 21 +3 -
C-R
3+1 8+7 -
24+9
C-L
2+2 7+4 2+2 23+9
analyzed histologically by light microscopy (Table II). As expected 5'24'37, the effectiveness of LPC in producing segmental demyelination in rat sciatic nerve was very high and was found in approx. 60% of all axons in both the C-L and R - L groups, but not in the C-R and R - R groups (Table II). The focal effect of L P C 24'37 w a s demonstrated by the lower frequency of segmental demyelination at the cuff region versus the injection region of the C-L and C-R groups (Table II). Paranodal demyelination also developed as a result of both the cuff operation and the injection into the right nerves of the R - R group and in all examined right (but not left) nerves (Table II), indicating that this phenomenon could be attributed primarily to the damage associated with the operation. However, in the injection region of the C-L and R-L groups, most axons had paranodal demyelination, and this lesion was less frequent in the C-R group. In contrast, in the cuff region, paranodal demyelination was most frequent in the C-R group, and there were fewer with paranodal demyelination in all the other groups. Degenerating axons were seen in both the cuff region and the injection region of the C-R and C-L groups (Table II). The degenerating axons were found mostly at the external perimeters of the nerve trunk, i.e. closer to the colchicine cuff. Thus, the degeneration could be attributed to the direct harmful effect of colchicine on survival of the axons. Degeneration was not found in the core axons, consistent with the finding of Jackson and Diamond 27. Degenerating profiles were rarely found in the R-L and R - R groups, indicating that the degeneration was not due to the cuff itself. Usually, a degenerating axon had paranodal demyelination, as previously demonstrated in many other cases 24'5°. An increase in axon diameter was found in the C-R and C-L groups at the cuff but not the injection region (Table II). The increased diameters were consistent with anticipated swelling of the axons at the regions where axonal transport is arrested 16'22'27. The implications for nerve conduction of all these histological changes will be discussed below. The results shown in Table II emphasize the different local effects at the cuff and injection regions. The left nerves were virtually unaffected. Electrophysiological measurements
Right nerve injection region Segmentaldemyelination 2+ 2 Paranodal demyelination 27 + 5 Axonal degeneration 5+ 4 Axonal diameter (pm) 22 + 5
58 +8"** 67 +9"** 9 +3 27 +9
15+8"* 30+7 30+5** 33+6
60+7"** 71+9"** 42+8*** 32+8
Right nerve cuff region Segmental demyelination 5 + 2 Paranodal demyelination 13 + 3 Axonal degeneration 3+ 4 Axonal diameter (pm) 24 + 3
4+ 3 25 + 7 7+ 4 23 _+7
12+6 35+3** 40 + 8 37+9
15_+7 30+4** 45 + 9*** 35+7
The influence of all treatments on nerve conduction was tested in parallel. Nerves removed within 1 h postoperatively did not conduct action potential (not shown). Two days postoperatively, recovery of conduction was seen in the R - R group. Eight days postoperatively, conduction was found in the R-R, R-L, and C-R groups. There was almost no recovery in the C-L group. The results, summarized in Table III, showed that the
53 TABLE III Electrophysiological parameters
Action potential amplitude (APA), action potential duration at 90% amplitude (APD9o), and conduction velocity (CV) were recorded at supramaximal stimulation. Current threshold at infinite stimulus (lr) was recorded from strength-duration curves. All measurements were made 8 days after cuff and injection were applied. Measurements are described for the cuff and injection regions (right nerve) and for the whole nerve (left nerve). Values are means + S.D. and were compared using a two-tailed unpaired t-test. *, P < 0.02; **, P < 0.002, versus R-R group in each right nerve. Parameter
R-R
R- L
C-R
C- L
APA (mV) Left whole nerve Right nerve cuff region Right nerve injection region
4.5 + 3.9 2.2 + 0.8 2.1 + 1.6
6.0 + 0.4 7.2 + 1.6" 4.4 + 1.8
4.0 + 3.5 0.6 + 0.2 1.5 + 1.3
5.2 + 0.6 1.1 + 0.4 0.4 + 0.3**
APDgo (ms) Left whole nerve Right nerve cuffregion Right nerve injection region
1.4 + 0.3 1.1 + 1.1 0.5 ___0.2
1.1 + 0.4 0.8 + 0.3 3.7 + 0.4**
0.9 + 0.3 0.6 + 0.3 0.6 __+0.4
0.5 + 0.3 0.6 + 0.1 0.7 + 0.3
c v (m/s) Left whole nerve Right nerve cuffregion Right nerve injection region
50.5 + 24.7 43.6 + 21.8 49.1 + 3.1
56.7 + 15.8 29.1 + 5.9**
45.0 + 4.5 53.8 + 15.0 50.0 + 5.0
37.5 + 17.7 31.1 + 16.7 37.5 + 17.7
I~ (gA) Left whole nerve Right nerve cuffregion Right nerve injection region
150 _+77 92 _+30 139 + 42
187 _+25 251 _+ 180 245 _+7**
161 + 155 160 _+40
185 _+78 156 + 114 194 _+37
p a r a m e t e r s of the left nerves in all groups were similar, regardless of the t r e a t m e n t , reinforcing our earlier impression from the behavioral and histological studies that t h e r e was no significant systemic effect of either L P C or colchicine on the m e a s u r e d parameters. T h e e x a m p l e s given in Fig. 1 A - D and the results s u m m a r i z e d in Table I I I show that with sham t r e a t m e n t ( R - R group), nerve conduction was partially i m p a i r e d , as indicated by reduction of the action potential a m p l i t u d e in the right versus left nerves of the R - R group (Table III). The degree o f injury induced by the surgical m a n i p u l a t i o n was increased from proximal to distal (Fig. 1A, Table III). In view of all the preceding, in each s e g m e n t all p a r a m e t e r s in each t r e a t e d group were c o m p a r e d to those of the corresponding segment of the right nerves o f the R - R group and not to those of the left nerves of the t r e a t e d group. T h e impact of L P C d e m y e l i n a t i o n is shown in Fig. 1B,C and Table III. Two days after L P C injection, there was practically no conduction through the LPC injection s e g m e n t (electrodes 3 - 4 , Fig. 1B). Eight days after the o p e r a t i o n , nerve conduction was r e s t o r e d (Fig. 1C). P r o p a g a t i o n through the d e m y e l i n a t e d region had a significantly low conduction velocity, and the threshold current (It) was significantly higher. These results are u n d e r s t a n d a b l e if one assumes that the conducting axons either are n o n - m y e l i n a t e d or have short-segmented internodes 19'24'a5'44. In either case, nerve conduction is antic i p a t e d to be slower 18'26'52. A l s o consistent with this
assumption was the finding of an action potential with two peaks, a larger and a smaller one (electrodes 3 - 4 , Fig. 1C). The first c o r r e s p o n d e d to the impulse g e n e r a t e d close to the recording e l e c t r o d e across the d e m y e l i n a t e d region, while the second, smaller one could be an ectopic one g e n e r a t e d at the d e m y e l i n a t e d segment, which, due to i m p e d a n c e mismatch 29'52, could be p r o p a g a t e d electrotonically. Such records have been p r e d i c t e d in simulation analysis of nerve conduction at d e m y e l i n a t i o n as described using various m a t h e m a t i c a l m o d e l s 28'29'41"52. The presence of a colchicine cuff proximal to the L P C - i n d u c e d focal d e m y e l i n a t i o n (C-L group) p r e v e n t e d the recovery of both action potential a m p l i t u d e and duration at the L P C - i n j e c t e d region (Fig. 1D, Table III). The tiny action potential d e t e c t e d in these circumstances had a normal conduction velocity, suggesting that the conducting axons were the few that were not injured by L P C and therefore c o n d u c t e d the typical saltatory and rapid action potentials 18'26. Recording at the injection region showed that the colchicine cuff was harmful in itself (C-R group, Table III), as demonstrated by the reduction in action potential amplitude and duration in the C - R versus R - R groups. However, conduction velocity and threshold currents in the C-R group were similar to those in the R - R group. These results suggest that, although colchicine induced degeneration of a substantial number of axons, the injured axons degenerated and simply disappeared from the records, while the remaining ones were more or less normal.
54
4k
150.0-
,
R-R
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,
I
90.0060.00-
-60.000
~-~30.00~
1oool
-30.000 .
.
.
.
.•o• 150.0 -
.
R-L
II~
1.0000
I
C_L _400.0
90.00
~. 60.00 ,u
. .o. .o. ., v o
~ 30.00 m 1.000 0
30°.0
-200.0 10
~ ~J 3'0
40
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- 100.0
i.000 i0 20 30 40 50 Distance from spinal cord (mini
Fig. 2. Binding of mAb SC-72-38 to sciatic nerves. Pooled values of left nerves are shown for each group (see text). Arrows indicate cuff (left) and injection (right) regions. Lower panel: schematic diagram of surgical lesion with respect to the different segments. It is interesting to compare the changes in action potential parameters between the injection and cuff regions. In the latter, all action potential parameters of the R-L group were larger than in the R-R group, except for nerve conduction velocity, which was significantly slower. These results can be understood using the prediction made by the mathematical models of impulse propagation from myelinated to demyelinated segments 4' 1 3 , 1 5 , 2 8 , 2 9 , 4 1 , 5 2 . These models predict that at the segments preceding the demyelinated segments, the propagating action potential would have a slower conduction, since the cable space constant is influenced by the increased capacity of the membrane in the demyelinated segments ahead 28'29'52. Also, if the demyelinated region recruits more sodium channels, the impulse at the preceding segments would be expected to be larger 4'15'29'41"52. The increased duration should be anticipated, considering that the rate of rise of the action potential decreases as
TABLE IV Binding of anti-C 1÷ to sciatic nerve
Th binding of sodium channel-specific polyclonal antibodies generated against a synthetic peptide denoted C1÷ (anti-C1÷) was measured. Values were normalized to protein content determined by the Lowry procedure32. When more than one experiment was performed, values are the means + S.D. of 5 experiments. Nervesegment
R-R
R-L
C-R
C-L
Left whole nerve 262+240 250+31 300+180 250+171 Right nerve Proximalto cuff 73 + 41 200 1430 + 60 52,397 + 532 Cuffregion 202 + 53 261 + 14 26 + 11 46 Injection region 347 + 47 163 + 15 163 + 39 70 Distal to injection 98 + 30 162 + 36 28 + 13 75 + 27
a result of the capacitance increase at the demyelinated region15,28,29, 41" The increased duration and amplitude and the decreased conduction velocity at the cuff region were not seen in the C-L group. The differences between the R-L and C-L groups were consistent with the failures in impulse propagation through the demyelinated segment in the C-L group (Table III). The widening of the axons at the cuff region in the C-L group adds to the decreased safety factor for impulse propagation from the preceding into the demyelinated region 4'20'29,39,41. The C-R group was less injured than the C-L group, indicating that the combination of axonal transport block and demyelination prevented recovery from the focal demyelination. B i n d i n g studies
Involvement of sodium channel delivery from cell bodies to the LPC-injected region was demonstrated by measuring the number and distribution of sodium channels along the treated right and non-treated left nerves in all experimental groups. It was discovered that 8 days after injection of LPC, the number of binding sites to both mAb SC-72-38 (Fig. 2) and anti-C1 ÷ (Table (IV) at the LPC-injected segment of the R-L group was 2- to 4-fold larger than in the nerves of the R-R group or in the left, untreated nerves. These results confirm previous studies performed in our laboratory 35'37. The placement of a colchicine cuff between the cell bodies and the LPC-injected segment (C-L group) prevented the recruitment of sodium channel antigenic determinants at the demyelinated region (Fig. 2, Table IV). Accumulation of antibody-binding sites was found proximal to the colchicine cuff (C-R group) (Fig. 2, Table IV). In the C-L group, accumulation proximal to the colchicine cuff was greater than in the C-R group (Fig. 2, Table IV). Altogether, results of the binding experiments showed that at LPC demyelination of rat sciatic nerve, there was a focal increase in antigenic determinants associated with functional domains of the sodium channel. Channel recruitment could be prevented by the classical fast axonal transport blocker colchicine 12'21'27. The latter was equally efficient in blocking delivery of sodium channels to the rest of the axon, even without LPC-induced demyelination, confirming the central role of fast axonal transport in carrying sodium channels in uninjured nerves, as previously reported 31. From the results obtained in the C-L group, it appears that at demyelination more channels were being transported that way, as indicated by the larger accumulation in the C-L versus C-R groups. As the colchicine cuff prevented both the supply of sodium channel antigenic determinants and the recovery of function at demyelination, the two processes
55 are probably interrelated. Thus, our results can serve to emphasize the central role of sodium channel delivery by axoplasmic transport in functional recovery from demyelination. DISCUSSION An increase in sodium channels at demyelination of mammalian nerves has been implied in many studies 5' 19,35,37,40,42,48,51. A somewhat different observation was made in frog sciatic nerve, in which LPC was found to be capable of inducing focal demyelination 46. However, when the loose patch-clamp technique was applied to recording sodium currents at the demyelinated axons, the detected current density in nodes corresponded to 10002000 sites//~m2 (refs. 43, 46), and at the demyelinated axons the current density corresponded to 60-100 channels//~m2. In the demyelinated region, the sodium channel density immediately after demyelination and during the next 6 days did not change 46. In parallel, impulse propagation through the demyelinated region was not restored during the first 6 days 46. The differences between frog and rat in regard to recovery from demyelination can be attributed simply to the differences in species and in the measuring techniques. However, they can also be attributed to the difference in body temperature between frog (4-20 °C) and rat (37 °C). It is well known that fast axonal transport is slowed by cooling from 37 to 20 °C and is completely arrested at 4 °C x3'22. Thus, the lack of increase in sodium channels and the failure in recovery of function at demyelinated axons of the frog 8 days postoperatively may be a result of the lower efficiency of fast axonal transport in delivering sodium channels in frogs versus rats. A comparative study should be performed to examine this assumption. There is one conceptual concern with our findings with regard to specificity of coichicine action, especially on a long-term scale. To study the long-term fate of the REFERENCES 1 Allen, R.D., Weiss, D.G., Hayden, J.H., Brown, D.T. and Fugiwake, H., Gliding movement and bidirectional transport along single native microtubules from squid axoplasm: evidence for an active role of microtubules in cytoplasm transport, J. Cell Biol., 100 (1985) 1736-1752. 2 Angelides, K.J., Elmer, L.W., Loftus, D. and Elson, E., Distribution and lateral mobility of voltage-dependent sodium channels in neurons, J. Cell Biol., 106 (1988) 1911-1925. 3 Barhanin, J., Meiri, H., Romey, G., Pauron, D. and Lazdunski, M., A monoclonal immunotoxin acting on the Na ÷ channel, with properties similar to those of a scorpion toxin, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 1842-1846. 4 Berkinblit, M.B., Vvedenkaya, N.D., Guedenko, L.S., Kovalev, S.A., Kholopov, A.V., Fomin, S.V. and Chailakhyan, L.M., Computer investigation of the features of conduction of a nerve impulse along fibres with different degrees of widening, Bio-
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