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Remyelination and recovery of conduction in cat optic nerve after demyelination by pressure
Experimental Neurology 184 (2003) 865 – 877 www.elsevier.com/locate/yexnr
Remyelination and recovery of conduction in cat optic nerve after demyelination by pressure Lynne J. Cottee, a C. Daniel, b Wai Sim Loh, a B.M. Harrison, c and W. Burke a,b,* a
b
Department of Physiology, Institute for Biomedical Research, University of Sydney, Australia Department of Anatomy/Histology, Institute for Biomedical Research, University of Sydney, Australia c Department of Biological Sciences, University of Technology Sydney, Australia Received 20 April 2000; revised 11 June 2003; accepted 13 June 2003
Abstract Pressure has been applied to the optic nerve of cats sufficient to block conduction in the large (Y) nerve fibers. The pressure block produces a mixture of axotomy and demyelination. By means of implanted electrodes, recovery of conduction in these fibers was monitored. There is a short-term recovery starting about 2 weeks after block induction and finishing at about 4 weeks. A later recovery starts at about 6 – 7 weeks and finishes at about 10 – 11 weeks. The remyelination has been monitored in the electron microscope by measurement of the myelin thickness and axon diameter of the large fibers. The remyelination follows a time course similar to the late phase of conduction recovery. By reference to the work of others, we surmise that the early recovery of conduction is due to the reorganization of microtubules disorganized by the pressure. D 2003 Elsevier Inc. All rights reserved. Keywords: Demyelination; Remyelination; Conduction recovery; Microtubules; Optic nerve; Cat
Introduction Demyelination causes a block in impulse conduction because the demyelinated axon membrane lacks an adequate regenerative ionic mechanism (Ritchie and Rogart, 1977). At a later time, sodium channels develop (Foster et al., 1980) and conduction again becomes possible, albeit now at very low conduction velocity, either continuous or saltatory (Bostock and Sears, 1978). Overlapping this process in time and eventually superseding it is the process of remyelination, which leads to a restoration of normal conduction. The exact relationship between the recovery of normal conduction and the remyelination is not clear. There are comparatively few reports on the time course of remyelination or the time course of restoration of conduction and no quantitative study on the two time courses in the same tissue. These remarks apply as much to the central nervous system (CNS) as to the peripheral nervous system. Time courses of remyelination in the central nervous system have
* Corresponding author. Department of Physiology, University of Sydney, NSW 2006, Australia. Fax: +61-2-9351-2058. E-mail address: [email protected] (W. Burke). 0014-4886/03/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0014-4886(03)00310-8
been described in (or can be inferred from) several reports (Clifford-Jones et al., 1980; Gledhill and McDonald, 1977; Gledhill et al., 1973; Harrison and McDonald, 1977; Hildebrand et al., 1985; Ludwin and Maitland, 1984; O’Leary and Blakemore, 1997; Smith et al., 1981). Reports of time courses of recovery of conduction are fewer (Felts and Smith, 1992; Fowler et al., 1972; Smith and Hall, 1980; Smith et al., 1981). In only one of these reports is it possible to compare the two time courses on neurons in the CNS. The paper of Smith et al. (1981) describes recovery of conduction in the spinal cord of the cat following demyelination by lysophosphatidylcholine; the recovery of conduction is not graphed and the remyelination is described only qualitatively. There have also been descriptions of recovery of function (Blight, 1993; Gledhill et al., 1973; Jacobson et al., 1979). One of these papers (Gledhill et al., 1973) also describes recovery of myelination. In the present paper, we give quantitative descriptions of the time courses of remyelination and of recovery of conduction in the same tissue, the optic nerve of the cat. Demyelination can be produced by pressure, by chemicals and by disease. The time courses of recovery may be different depending on the demyelinating agent and on the
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persistence of the agent. The advantage of using pressure is that once pressure has been withdrawn, all subsequent effects are due only to the initial pressure and are not dependent on any continued action by the agent. Selective pressure has been applied to the optic nerve of the cat with the intention of blocking conduction in the large (Y) fibers. The main purpose of this procedure was to elucidate the function of the Y fibers by comparing the effects of stimulating the Y-blocked nerve with the effects of stimulating the normal nerve on binocular cells in the brain (reviewed in Burke et al., 1998). When pressure is applied to a nerve, conduction in individual fibers may be blocked in at least five ways: (i) by hypoxia, (ii) by depolarization caused by accumulation of potassium ions and possibly other metabolites and by entry of calcium into the axoplasm, (iii) by disruption of the cytoskeleton, (iv) by demyelination and (v) by axotomy. The first two causes are quickly reversible, within minutes or hours. Recovery from disruption of microtubules may take days (Kitao et al., 1997). Recovery from demyelination takes weeks (see papers cited above), while there is no recovery from axotomy in the CNS unless special measures are taken (reviewed in Stichel and Mu¨ller, 1998). In our experiments on cats, we have applied pressure of sufficient magnitude and duration to produce a mixture of demyelination and axotomy of the large (Y) fibers of the optic nerve. In the electron microscope, the axotomised (degenerating) fibers are readily distinguishable from the demyelinated (or remyelinating) fibers. Hence, we were able to measure axon diameter and myelin thickness in the latter fibers at various times after the pressure block. In the same cats, with implanted electrodes, we could stimulate and record from optic nerve or tract and therefore could monitor any recovery of conduction in the Y fibers. The two time courses (morphological and functional) are different and a reason for this will be suggested.
Methods Surgical operation Cats were anaesthetized with sodium pentobarbitone, 40 mg/kg ip and as needed to maintain surgical anaesthesia. All operations were conducted with sterile or aseptic precautions. Bipolar stainless steel electrodes were inserted into optic tract (Fig. 1A, position 3) and intracranial optic nerve (Fig. 1A, position 2) using the response to a bright flash as a guide to position. One optic nerve (usually the left) was exposed by a dorsal approach. Fine enamelled stainless steel wire electrodes, the ends sharpened and bent back 1 – 2 mm, were hooked into the connective tissue adjacent to the junction of the nerve and eyeball (position 1) and cemented in place with cyanoacrylate glue. Using a special cuff 2 –3 mm wide (see Burke et al., 1986 for a full description) positioned a few millimeters
from the eyeball (Fig. 1A), we applied pressure to the exposed optic nerve sufficient to block conduction in the large (Y) fibers. Impulse conduction through the pressurized region was monitored by stimulating at electrodes 2 or 3 while recording the response (the compound action potential) from electrode 1. We applied pressure for no more than 30 s, then waited 1– 2 min before reapplying pressure. By starting at a low pressure (100 kPa) and gradually increasing the pressure with each successive application, it was possible to reduce the t1 response (the response of the Y fibers; Fig. 1B) with little or no effect on the t2 response (the response of the X fibers; Fig. 1B). We aimed to reduce the t1 response to less than 10%, without reducing the t2 response below 90%. Pressures near 200 kPa were usually needed to achieve this. When this had been done, the pressure device was withdrawn, the electrodes soldered to a 9-pin plug that was then firmly attached to the skull by dental acrylic, and the skin sutured. Buprenorphine, a long-lasting anaesthetic, 0.03 mg im, was given then and at 12-h intervals as required. Subsequently, the optic tract or nerve was stimulated and recorded from via the plug. Further details are given in previous publications (Burke et al., 1986, 1987, 1992, 1998; Cottee et al., 1991). Monitoring of responses Following the operation, we monitored the responses, at first daily, later at 2- to 3-day intervals, using single shocks to the intracranial optic nerve or tract. Our usual stimuli were 3 mA 0.05 ms; such stimuli produce a good but not maximal response. They do not produce aversive behaviour in the unrestrained animal, indeed may not even be perceived. By stimulating at electrodes 2 or 3 and recording the compound action potential from electrode 1, we determined whether the pressure block was maintained or whether there was any recovery of response. We also recorded from electrode 2 in response to stimulation at position 3 (or vice-versa) and used this response (Fig. 1C) to determine whether some fibers had been axotomised and ceased to conduct into the optic tract (see Results for a full description). Histology The aim of the histological procedures was to determine the myelin thickness in axons of various diameters. The blocked optic nerves were examined histologically at various times after the initial operation. Six normal nerves, either from pressure-blocked cats or unoperated cats, were also examined. The cats were deeply anaesthetized with sodium pentobarbitone (80 mg/kg ip) and respired with carbogen (95% O2, 5% CO2). The descending aorta was clamped and 5 ml of 1% sodium nitrite solution containing 500 IU heparin was injected into the left ventricle. One minute later, the head was rapidly perfused with 300 ml of warm (37 –40jC) 0.9% NaCl solution at pH 7.4. This was
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Fig. 1. Pressure block of cat optic nerve. (A) Diagram of peripheral visual system, to show eyes, optic nerves, optic chiasm, optic tracts and lateral geniculate nuclei (LGN). Electrodes are placed in right optic tract (ROT; position 3), left intracranial optic nerve (LICON; position 2) and in the left extracranial optic nerve (LECON) near the eyeball (position 1). Pressure is applied in the region 5 – 8 mm from the eyeball (shaded). Scale approximate. (B) Response at position 1 to stimulation at position 3 before the block (dark trace); after the block, the t1 response is virtually absent, while the t2 response is slightly reduced and delayed (faint trace) (cat LB165). (C) Response at position 3 to stimulation at position 2 one day after the block (dark trace); 4 days after the block, the t1 response has decreased to about 10%, while the t2 response is unchanged (faint trace) (cat LB152). In (B) and (C), negativity is upwards; the amplitudes of all responses were measured as positive peak to negative peak; from 5 to 32 responses were averaged for each datum point. (D) Amplitude of the t1 response recorded as in (C) as a function of time after pressure block. Note the sharp decline between 100 and 200 h (cat LB40).
followed by about 3 l of 0.1 M phosphate buffer containing 4% paraformaldehyde, 0.5% glutaraldehyde and 0.54% dextrose, adjusted to pH 7.4, and perfused over about 30 min, initially at a pressure of about 20 kPa, later at 2 –5 kPa. The nerves were dissected out 1– 2 h later and placed in a fixative solution the same as the above except for the glutaraldehyde concentration that was increased to 1%.
After overnight fixation at 5jC, the nerves were cut transversely into pieces 0.4 mm long using a vibratome, further fixed in 1% osmium tetroxide and then embedded in epoxy resin (Durcupan ACM, Fluka). Cross-sections of the nerves were taken from the pressurized regions or, in the case of the normal nerves, from a corresponding part of the nerve. Complete cross-
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sections cut at 1-Am thickness and stained with 0.25% toluidine blue in 0.5% dimethylaminoethanol were viewed in the light microscope to check for adequate fixation. Sections across one diameter of the nerve were cut at 70– 80 nm, stained with uranyl acetate and lead citrate (Reynolds, 1963) and viewed in a Philips CM12 microscope. Images were taken across the entire diameter at magnifications of between 5600 and 8800 times. These images were saved directly onto the hard drive of a PowerMac for later analysis. It was not our intention to measure all axons in a section since we were not aiming to obtain a population distribution. Because the pressure-blocking operation affected primarily the axons with diameters more than 5 Am, we concentrated on these. We adopted the value of 5 Am as criterion for the lower limit of Y fiber diameter and we have assumed that the t1 response (see later) is the response of the Y fibers (see Burke et al., 1986 and Cottee et al., 1991 for a discussion of these points). However, for purposes of comparison, a wide sprinkling of smaller axons was measured. Any axonal profiles that were degenerating or grossly distorted were omitted. Myelin thickness was measured on a compact part of the myelin. The axon perimeter and myelin thickness were measured using the PrismView data analysis program (Analytical Vision Inc., Raleigh, NC, USA); this program also calculated axon area. For the magnifications used, the limit of resolution (one pixel) was 0.010 – 0.016 Am. Axon diameter was determined in two ways. First, the diameter was taken to be the perimeter divided by p; this assumes that the length of the perimeter is not changed by the blocking procedure and that the axon usually has a circular profile. The plane of section of the nerves was orthogonal to their long axes. Of course, individual fibers can run obliquely but no correction was made for such fibers. It has been shown that axotomised axons in the peripheral nervous system tend to retain their circumference while losing volume, becoming crenated (Gillespie and Stein, 1983); we assume the same is true for injured central axons. Axon diameter was also calculated as the diameter of a circle with the same area as that measured. This assumes that the area as measured is the same as in life; this method gives slightly smaller diameters than the first method. We determined axon diameter by both methods for most experiments. However, the results shown in all the figures are based on the first method. The results were qualitatively the same for both methods. For statistical comparisons between groups, we used the Mann – Whitney U test; to test the significance of differences between slopes of regression lines, we used a t test (Goldstein, 1964). We adopted P V 0.05 for statistical significance. All procedures were carried out in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and approved by the Animal Care Ethics Committee of the University of Sydney.
Fig. 2. Recovery of t1 response in pressure-blocked optic nerve. (A) Response recorded as in Fig. 1B at various times after the block. Note the gradual recovery over about 5 weeks (cat LB158). (B) Graph of the changes in the t1 and t2 responses in a pressure-blocked optic nerve recorded as in Fig. 1B over about 7 weeks. Note the slow decline in the t2 response but recovery in the t1 response (cat LB40).
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Fig. 3. Graphs of the change in the t1 response in five pressure-blocked optic nerves. Recording as in Fig. 2 and normalization both to 0% (the weakest response post-block) and to 100% (final steady level). Note that four nerves show a two-stage recovery whereas one shows only the late recovery. One response for cat LB152 at 166 days has been omitted.
Fig. 4. Recovery of response in pressure-blocked nerves. The continuous solid line is the mean of the separate graphs in Fig. 3, interpolating where appropriate; averages were taken only when at least three values were available. The individual points and dashed line were obtained from measurements made (by us) in Fig. 5 of the paper of Smith et al. (1981), normalizing both to 0% (minimum response) and to a steady level at 100%. (In the data of Smith et al., there appeared to be a recovery to about 50% of the pre-block level.) Note the general resemblance of the two graphs.
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Results Degeneration versus demyelination Fig. 1A shows a diagram of the optic nerves, optic chiasm and optic tracts in the cat. Pressure is applied to one nerve close to the eyeball (shaded region) while monitoring conduction through this region by stimulating chiasm or tract at electrodes 2 or 3 and recording from an electrode at 1. A conduction block of the t1 response (the response of the Y fibers) is considered satisfactory when this response drops out leaving only the t2 response (the response of the X fibers): compare the responses in Fig. 1B. At this time, the responses from electrode 2 to stimulation at
electrode 3 (or vice-versa) show both the t1 and t2 responses (Fig. 1C) because the axons running between these two electrodes have normal conduction properties. However, if any axons have been axotomised by the pressure, their axons in this region will eventually lose membrane potential and cease to conduct. For Y fibers, this happens fairly abruptly on about the fourth day and the t1 response decreases (compare the responses in Fig. 1C; see Burke et al., 1986 for a full description). Note that when electrode 2 is in the chiasm, the response includes the response of some fibers not subjected to pressure block, that is, those fibers travelling ipsilaterally from retina to tract. To monitor only fibers travelling through the block site, it is necessary to place electrode 2 in the intracranial optic nerve; the elec-
Fig. 5. Electron microscopical appearance of normal optic nerve axons and axons subjected to pressure block. (A) Large axon in normal optic nerve. (B) Completely demyelinated medium-sized axon 27 days after pressure block (cat LB61). (C) Demyelinated – partly remyelinated large axon 53 days after pressure block (cat LB40). (D) Demyelinated – partly remyelinated large axon 53 days after pressure block (cat LB40). Note the extremely attenuated cytoplasm and the crenated myelin sheath. Scale bar (2 Am) applies to all sections.
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trode is satisfactorily placed when it records no response to stimulation of the contralateral eye. Depending on the particular circumstances attending the application of pressure, there may be a complete or only partial loss of t1 response in the region caudal to the block site (Fig. 1C). Fig. 1D exemplifies a case of large but nontotal loss of t1 response. The electrophysiological finding of a loss of t1 response at about 4 days is indicative of axotomy, a conclusion supported by morphological evidence of degeneration (Burke et al., 1986). It seems to be impossible to obtain a 90 – 100% t1 conduction block in the optic nerve without some, often considerable, degeneration. The conduction block in the nerve is maintained for at least a week, and often for several weeks, but usually there is some recovery of the t1 response, the extent of which depends on the degree of loss at 4 days (Figs. 1C, D); the greater the loss at 4 days, the less chance of recovery later. Recovery of t1 response Fig. 2A shows compound action potentials recorded from the optic nerve at electrode 1 in response to stimulation of the optic tract via electrode 3 at various times after a pressure block in one cat (LB158). Note that the t1 response appears at its normal latency (e.g. trace d). It is possible that during the process of recovery, individual Y axons may discharge at longer latencies, perhaps during the t2 response,
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but such discharges would probably be very asynchronous and could not be reliably detected. In Fig. 2B is the graph of recovery of the t1 response in another cat (LB40). Recovery commenced at about 20 days and reached a peak about 8 days later; there then seemed to be a later phase of recovery at about 42– 48 days. At the same time, there was only a decrease in the t2 response, a common occurrence. Fig. 3 graphs the t1 recovery curves in five cats. In all cases the responses have been normalized, to 0% for the smallest post-block response and to 100% for the final roughly steady level. Although there is considerable variability in the time courses, four of the five graphs reach 100% in about 30 days. Fig. 4 is a graph of the mean of the graphs in Fig. 3 (solid line). This emphasizes the two-stage recovery. To the graph of Fig. 4, we have added measurements we made on records of recovery of responses in the spinal cord of a cat treated with lysophosphatidylcholine (Fig. 5 in Smith et al., 1981; the short-latency response measured peak-to-peak; dashed line). The similarity of the two graphs will be considered in the Discussion. Remyelination Fig. 5 shows electron micrographs of a large axon in a normal optic nerve (Fig. 5A) and in nerves at 27 days (Fig.
Fig. 6. Relation of myelin thickness to axon diameter. (A) Plot of myelin thickness versus axon diameter for six normal optic nerves (data combined: 2216 axons), with regression line (solid line). The dashed lines are the regression lines for the 0- to 3-Am and >5-Am diameter axons; note the differences in their slopes from that of the solid regression line. (B) Plot of the myelin thickness/axon diameter (MT/AD) ratio for groups of axons of different diameters, with regression line. Points plotted at the median position of the group; SEMs plotted but smaller than the symbols in most cases.
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5B) and 53 days (Figs. 5C, D) after the pressure block. There is complete demyelination in the medium-sized axon shown in Fig. 5B at 27 days. Complete demyelination has been seen in other axons of similar size at this interval; the large axons had presumably either retained or reacquired myelin. At 53 days (Fig. 5C), remyelination was under way. In some axons at 53 days (Fig. 5D), the axoplasm appeared almost featureless and yet the axons did not appear to be degenerating; they may represent axons from which the axoplasm has been extruded by the pressure but without injury to the plasma membrane. In the fiber of Fig. 5D, the myelin appears crenated. On the assumption that the recovery of conduction was mainly due to remyelination, we attempted to substantiate
this idea by measuring axon diameters and myelin thicknesses at various times after the pressure block. Of course, in contradistinction to the electrophysiological measurements, we could not make serial measurements in a single animal but only a single measurement at a particular interval. We did not attempt to distinguish between partly demyelinated and remyelinating fibers. For comparison, we made measurements on six normal optic nerves. The data from all six nerves have been combined in a single graph in Fig. 6A. A regression line (solid line) gives a reasonable fit to the data, in agreement with several previous reports on both optic nerve (Bishop et al., 1971; Friede et al., 1971; Williams and Chalupa, 1983) and other tissues (Ludwin and Maitland, 1984; O’Leary and
Fig. 7. Plots of myelin thickness versus axon diameter for six pressure-blocked nerves. Data obtained at the post-block intervals indicated. Note that the slopes of the regression lines for axons of 0- to 3-Am diameter remain similar to those in normal nerves, whereas for axons >5-Am diameter, the slopes are radically changed (9 days, cat LB39; 27 days, cat LB61; 53 days, cat LB40; 79 days, cat LB158; 97 days, cat LB57; 167 days, cat LB152).
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Blakemore, 1997). Since our primary interest was in axons >5-Am diameter, we have added the regression line (dashed) for these axons, and for comparison, the regression line (dashed) for axons 0- to 3-Am diameter. The 0- to 3-Am group was chosen for comparison because the pressure block may affect some fibers with axon diameters less than 5 Am, but probably none with diameters less than 3 Am. It is evident that the single regression line is only an approximation for the whole population. The relation between myelin thickness and axon diameter is commonly depicted by graphing axon diameter against fiber diameter. The slope of this graph is the ‘g’ ratio and, in our terms, equals AD / (AD + 2MT). For cat optic nerve, Williams and Chalupa (1983) reported a value of 0.8 for the g ratio, while from Fig. 6A, this ratio is found to be 0.82. The MT/AD ratio is a simpler way of expressing the relationship and is used throughout this paper. It may be noted, however, that the MT/AD ratio is not constant but decreases with increase in axon size (Fig. 6B). It follows that the g ratio is likewise not constant but increases in the larger fibers, as has been observed previously for peripheral nerve with light microscopy (e.g. Sanders, 1948), but is not usually seen with the electron microscope (reviewed in Waxman and Swadlow, 1977). Fig. 7 shows graphs for myelin thickness versus axon diameter for various times after a pressure block with separate regression lines for the 0- to 3-Am group and the >5-Am group. It seems that the 0- to 3-Am group is essentially unchanged from normal at most intervals (no data were
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collected at 79 days); however, in the >5-Am group, the regression line has a much reduced slope, even becoming negative at 27 days but then increasing at longer intervals. Although the change in slope of the regression line gives an approximate measure of the time course of remyelination, this is not a good measure for several reasons. The regression line does not necessarily go through the origin. Also, if sampling is not extensive and does not include an adequate selection of fibers, the regression line will be distorted. A better way is to determine the myelin thickness/axon diameter (MT/AD) ratio for individual fibers and to compare these values with the corresponding ratios for normal fibers. It is necessary to make the comparison with the appropriate group of normal axons because of the decrease in this ratio with increase of axon diameter (Fig. 6B). The MT/AD ratios for diameter subgroups >5 Am are plotted against time after pressure block in Fig. 8. Each datum point is the mean MT/AD ratio in the pressureblocked nerve for a particular axon diameter range (e.g. 5 –6 Am) expressed as a percentage of this ratio for axons of the same size in the normal nerve. All the curves show a similar time course. The decrease to a trough at 27 days is interpreted as evidence of progressive demyelination. However, remyelination can commence within 2 weeks of a lesion (Smith et al., 1981). Hence, during this period, both demyelination and remyelination are occurring. Recovery of myelin thickness to normal occurs at about 70 days and is followed by a period when the MT/AD ratio exceeds normal at 79 and 97 days. Only the 5- to 6-Am group (at both days)
Fig. 8. Recovery of myelin thickness – axon diameter relationship. For axons >5-Am diameter, graph of the MT/AD ratio for five subgroups expressed as percentages of this ratio in the corresponding normal groups against time after pressure block. The separate graphs for axons of different diameter follow a similar time course to one another. For the points at 79 and 97 days, only those for the 5- to 6-Am group are significantly above the normal level (100%).
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Fig. 9. Comparison of recovery of conduction with recovery of MT/AD ratio. Graph of the MT/AD ratio for axons >5-Am diameter versus time after pressure block (symbols and left scale). All points except that at 167 days are significantly different from the normal value ( P < 0.0001 except for 79 days where P < 0.005). For all points, the SEMs are smaller than the symbol; the SEM for the ratio for normal axons is similar. For comparison, the mean response recovery curve from Fig. 4 is reproduced here (continuous line and right scale). The MT/AD ratio may also be read as a percentage using the right scale.
is significantly above normal ( P < 0.005), but this statistical significance and the similar pattern of the other groups suggest that this is a genuine effect. At these times, there is either a hyper-myelination or a relative shrinkage of the axon. All the curves fall to normal myelin thickness by about 170 days. The time course of remyelination is made clearer by combining the data in Fig. 8 and plotting the means in Fig. 9; all points are significantly different from normal except that at 167 days. Also plotted in Fig. 9 is the mean response recovery curve reproduced from Fig. 4. The two recovery curves are similar from about 50 days but differ considerably before that. There is an early phase of functional recovery commencing at 1 –2 weeks and peaking at about 4 weeks. At the critical time of around 30 days, the two curves are significantly different ( P = 0.04: Mann –Whitney, two-tailed). If the result from cat LB152 is excluded (this cat did not show the early phase of recovery; cf. Fig. 3), the difference is even more significant ( P = 0.006).
Discussion Method of pressure block The cuff we used was up to 3 mm wide when inflated. It was shown by Gilliatt et al. (Fowler et al., 1972; Ochoa et al., 1972) that pressure with a pneumatic cuff tends to produce a movement of myelin away from the centre of the cuff towards the uncompressed part of the nerve result-
ing in an invagination of the myelin at several nodes. From their data, we can safely assume that all the nodes under our cuff (2 – 4) were affected. It appears that the myelin that is stretched may be separated from its Schwann cell (in Gilliatt’s experiments) and degenerate, resulting in a ‘paranodal’ demyelination. The Schwann cell divides, its daughter cell(s) then proceed to remyelinate the demyelinated paranodal region to form new short ‘intercalated’ internodes (Lubinska, 1958, 1961). If this process also occurs in central axons, it would result in a mixture of relatively normal myelin and myelin in various stages of demyelination/ remyelination. Our sections would be expected to sample in both regions. This might account for the large scatter of myelin thicknesses observed at all intervals after the block (see Fig. 7). Although this means the experiment is less than ideal, provided we are always sampling an adequate number of fibers, we should still be able to chart the time course of remyelination accurately. In the present samples, we did not encounter a single large fiber that was completely demyelinated. This may have been due to sampling at intervals when axons had not completely demyelinated (9 days) or had remyelinated to a significant extent (27 days onwards). It seems that the medium-sized fibers are differently affected by the pressure, many showing complete demyelination at 27 days (Fig. 5C). Recovery of conduction When considering the reasons for recovery of conduction in fibers pressure-blocked in the present experiments, we
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can dismiss re-oxygenation and dispersal of potassium and other metabolites because the block persists for at least 10 days and recovery from these causes should be a matter of hours. On the other hand, axotomy would lead to degeneration of the caudal segment of the axons and would rule out any recovery of conduction in those fibers. The two remaining recovery processes (restoration of the cytoskeleton and remyelination) are both candidates in our experiments. Can recovery of conduction be due to remyelination? If we compare the time courses of recovery of conduction and remyelination in our experiments, we see that they are similar from about 50 days but that the early parts of the time courses are very different (Fig. 9). First, at 9 days, there is almost complete block of conduction, yet the myelin thickness is about 83% of normal, suggesting that the cause of the block is not simply demyelination. Secondly, at about 30 days, in most cases there is complete recovery of conduction (see Fig. 3) whereas there is now a significant amount of demyelination. Subsequently, a partial conduction block is reestablished transiently despite some further remyelination at this time, again suggesting another process. It is interesting that a similar delay in recovery of conduction is seen in the results of Smith et al. (1981) from the cat spinal cord demyelinated by lysophosphatidylcholine (see Fig. 4). Against these rather slow changes in conduction and remyelination, it should be emphasized that the initial block of conduction was achieved within a few minutes. This is more likely to be due to movement of axoplasm and stretching of the myelin sheath [leading to invagination of the myelin (Ochoa et al., 1972), and at greater pressures, rupture of the plasma membrane] than to stripping of myelin that, as Gledhill et al. (1973) found, continues to occur over at least a week. There are at least two possible explanations for the early recovery of function. The first is that the conduction block is due to a detachment of the myelin loops at the paranodes without detachment or loss of myelin elsewhere. The axon – glia connection at the paranodes (the ‘transverse bands’) is considered to be labile (Waxman, 1981) yet is critical for conduction because it effectively separates the extracellular and periaxonal spaces (Hirano and Dembitzer, 1978), and its detachment causes a significant fall in transverse resistance at the internode (Funch and Faber, 1984) and also the exposure of potassium channels (Chiu and Ritchie, 1981), both events reducing the safety factor for transmission. By the same token, such a detachment might be quickly healed. A second possibility for the early recovery of function assumes that the pressure causes a disruption of the cytoskeleton of the axons, specifically the microtubules. It has been demonstrated that mechanical injury to axons can cause stretching of the plasma membrane leading to entry of calcium ions which, either alone or in association with calmodulin, cause disruption and loss of microtubules
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(reviewed in Maxwell et al., 1997). Recently, Hanke (2002) has shown that pressure applied to the optic nerve of rats causes disruption of heavy neurofilaments and failure of axonal transport both anterograde and retrograde, with restitution commencing at about 12 days. It has also been shown that pressure causing a disruption of microtubules can lead to a behavioural deficit (Kitao et al., 1997). Kitao et al. (1997) used pressures of 12.5 and 25 g/mm2 on rat tibial nerve; they demonstrated that the axons were not axotomised but that the microtubules were disrupted (more severely at the higher pressure) and that recovery, estimated behaviourally (recovery of normal gait), was complete at 3 days (lower pressure) and 21 days (higher pressure). These pressures are roughly equivalent to 125 and 250 kPa and so are similar to the pressures used in our experiments; we usually needed pressures of 180 –220 kPa to produce a block of conduction in the large (Y) optic fibers. It is therefore very probable that there was disruption of microtubules in our experiments. Of course, the simplest explanation for the experiments of Kitao et al. (1997) is that disruption of microtubules caused inadequate release of transmitter at the neuromuscular junction or at other synapses, with no need to suppose any alteration in nerve conduction. However, in a study of the effect of anoxia on nerve conduction by Waxman et al. (1992), it was suggested that in addition to the entry of calcium into the axoplasm (Ransom et al., 1990) and disruption of the cytoskeleton, the latter might also include effects on the proteins ankyrin and spectrin that are associated with the sodium channels (Srinivasan et al., 1988), and this might in turn contribute to the loss of conduction. This system recovers relatively quickly (Kitao et al., 1997). At the present time, it is not possible to decide between these two alternatives. Indeed, they are not strictly alternatives since they are not mutually exclusive. A variant of this explanation is that the pressure completely removes axoplasm from a length of fiber without damage to the plasma membrane, as has been shown can occur in the squid giant axon (Baker et al., 1962). If this happens, one would expect recovery to be fairly slow (see Fig. 5D). Late depression and recovery It is not clear why there is a decreased response between the early and late recoveries (Figs. 3, 4). One possibility is that there may be two phases of demyelination, the first, a paranodal detachment, discussed above, not clearly evident in the histology and rapidly reversed. The second phase of demyelination might be due to the loss of myelin by separation from the oligodendrocyte soma. An individual axon might exhibit only one phase of demyelination but the population of axons in one nerve could exhibit both phases. There seems to be a discrepancy between our results and those of Harrison and McDonald (1977). These authors
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applied pressure to the spinal cord of a cat for extended periods, up to 3 h. The weakness of the limbs resulting from this procedure had almost completely disappeared at 1 month (Gledhill et al., 1973), whereas remyelination was not complete even at 18 months (Harrison and McDonald, 1977). Possibly, the demyelination in the experiments of Harrison and McDonald (1977) was more extensive or more severe than in our experiments. It should be noted that our pressure block affects only (or mainly) the Y fibers which constitute less than 5% of the total population in the cat’s optic nerve. Thus, the environment of the remyelinating fibers in our experiments may have been much more favourable than in the experiments of Harrison and McDonald (1977). Similarly, Ludwin and Maitland (1984) found that remyelination after the use of cuprizone in mice failed markedly to reach normal myelin thickness levels even at 5 –6 months. Since this drug presumably affects all parts of an axon, full remyelination might take a considerable time. Hyper-myelination? The remyelinating fibers appear to undergo a period of hyper-myelination between 70 and 150 days (Fig. 8). This elevated MT/AD ratio does not seem to be artifactual because the pattern is repeated for each group of fibers (5 –6 Am, 6– 7 Am, etc.) and it remains statistically significant for the 5- to 6-Am group (Fig. 8). Both peripheral and central axons shrink when demyelinated. Prineas and Connell (1978) found that demyelinated axons in multiple sclerosis plaques had reduced axon diameters. Reduction in axon diameter has also been observed in demyelinated axons of the corpus callosum of mice on a diet containing cuprizone (Mason et al., 2001). If axons shrink on demyelination but recover their volume after about 100 days, this might account for the changes in the MT/AD ratio. We think this is unlikely because the elevated MT/AD ratio occurs when we calculate axon diameter from the measured perimeter by dividing by p, and because there is evidence that the perimeter does not change with injury (Gillespie and Stein, 1983; see also Fig. 5D). Thus, this method of calculation would be indifferent to changes in volume. It remains a possibility that the perimeter of an axon does not stay constant. It is interesting that Sanders (1948) also describes a hyper-myelination in the peroneal nerve of the rabbit following axotomy by crushing and observation in the light microscope. Both the proximal and distal segments show increase in myelin thickness, in spite of a decrease in axon diameter in the proximal stump and in the regenerating axons of the distal stump. The changes are greatest at 200 days after crushing and are reduced at 300 days. Sanders (1948) interpreted the increase in myelin thickness as due to a loss of tension within the axon. Although this idea would not find favour today, nevertheless, measurements of the periodicity of the myelin and the number of lamellae would clarify the phenomenon. Without further
information, we cannot decide between a hyper-myelination and changes in the axon. Acknowledgments This research was supported by grants from the National Health and Medical Research Council of Australia. We thank Ruz Djavadjian and Kris Turlejski for preliminary measurements and Bill Levick and Bogdan Dreher for invaluable comments on an earlier version of this manuscript. We thank the staff of the Electron Microscope Unit of the University of Sydney, particularly Dennis Dwarte, for their most helpful assistance. References Baker, P.F., Hodgkin, A.L., Shaw, T.I., 1962. Replacement of the axoplasm of giant nerve fibres with artificial solutions. J. Physiol. (London) 164, 330 – 354. Bishop, G.H., Clare, M.H., Landau, W.M., 1971. The relation of axon sheath thickness to fiber size in the central nervous system of vertebrates. Int. J. Neurosci. 2, 69 – 77. Blight, A.R., 1993. Remyelination, revascularization, and recovery of function in experimental spinal cord injury. In: Seil, F.J. (Ed.), Neural Injury and Regeneration. Raven Press, New York, pp. 91 – 104. Adv. Neurol. 59: 91 – 104. Bostock, H., Sears, T.A., 1978. The internodal axon membrane: electrical excitability and continuous conduction in segmental demyelination. J. Physiol. (London) 280, 273 – 301. Burke, W., Cottee, L.J., Garvey, J., Kumarasinghe, R., Kyriacou, C., 1986. Selective degeneration of optic nerve fibres in the cat produced by a pressure block. J. Physiol. (London) 376, 461 – 476. Burke, W., Cottee, L.J., Hamilton, K., Kerr, L., Kyriacou, C., Milosavlevic, M., 1987. Function of the Y optic nerve fibres in the cat: do they contribute to acuity and ability to discriminate fast motion? J. Physiol. (London) 392, 35 – 50. Burke, W., Dreher, B., Michalski, A., Cleland, B.G., Rowe, M.H., 1992. Effects of selective pressure block of Y-type optic nerve fibers on the receptive-field properties of neurons in the striate cortex of the cat. Vis. Neurosci. 9, 47 – 64. Burke, W., Dreher, B., Wang, C., 1998. Selective block of conduction in Y optic nerve fibres: significance for the concept of parallel processing. Eur. J. Neurosci. 10, 8 – 19. Chiu, S.Y., Ritchie, J.M., 1981. Evidence for the presence of potassium channels in the paranodal region of acutely demyelinated mammalian single nerve fibres. J. Physiol. (London) 313, 415 – 437. Clifford-Jones, R.E., Landon, D.N., McDonald, W.I., 1980. Remyelination during optic nerve compression. J. Neurol. Sci. 46, 239 – 243. Cottee, L.J., FitzGibbon, T., Westland, K., Burke, W., 1991. Long survival of retinal ganglion cells in the cat after selective crush of the optic nerve. Eur. J. Neurosci. 3, 1245 – 1254. Felts, P.A., Smith, K.J., 1992. Conduction properties of central nerve fibers remyelinated by Schwann cells. Brain Res. 574, 178 – 192. Foster, R.E., Whalen, C.C., Waxman, S.G., 1980. Reorganization of the axon membrane in demyelinated peripheral nerve fibers: morphological evidence. Science (Washington, DC) 210, 661 – 663. Fowler, T.J., Danta, G., Gilliatt, R.W., 1972. Recovery of nerve conduction after a pneumatic tourniquet: observations on the hind-limb of the baboon. J. Neurol. Neurosurg. Psychiatry 35, 638 – 647. Friede, R.L., Miyaghishi, T., Hu, K.H., 1971. Axon calibre, neurofilaments, microtubules, sheath thickness and cholesterol in cat optic nerve fibres. J. Anat. 108, 365 – 373.
L.J. Cottee et al. / Experimental Neurology 184 (2003) 865–877 Funch, P.G., Faber, D.S., 1984. Measurement of myelin sheath resistances: implications for axonal conduction and pathophysiology. Science (Washington, DC) 225, 538 – 540. Gillespie, M.J., Stein, R.B., 1983. The relationship between axon diameter, myelin thickness and conduction velocity during atrophy of mammalian peripheral nerves. Brain Res. 259, 41 – 56. Gledhill, R.F., McDonald, W.I., 1977. Morphological characteristics of central demyelination and remyelination: a single-fiber study. Ann. Neurol. 1, 552 – 560. Gledhill, R.F., Harrison, B.M., McDonald, W.I., 1973. Demyelination and remyelination after acute spinal cord compression. Exp. Neurol. 38, 472 – 487. Goldstein, A., 1964. Biostatistics. Macmillan, New York. Hanke, J., 2002. Anatomical correlations of intrinsic axon repair after partial optic nerve crush in rats. Ann. Anat. 184, 113 – 123. Harrison, B.M., McDonald, W.I., 1977. Remyelination after transient experimental compression of the spinal cord. Ann. Neurol. 1, 542 – 551. Hildebrand, C., Kocsis, J.D., Berglund, S., Waxman, S.G., 1985. Myelin sheath remodelling in regenerated rat sciatic nerve. Brain Res. 358, 163 – 170. Hirano, A., Dembitzer, H.M., 1978. Morphology of normal central myelinated axons. In: Waxman, S.G., Ritchie, J.M. (Eds.), Physiology and Pathobiology of Axons. Raven Press, New York, pp. 65 – 82. Jacobson, S.G., Eames, R.A., McDonald, W.I., 1979. Optic nerve fibre lesions in adult cats: pattern of recovery of spatial vision. Exp. Brain Res. 36, 491 – 508. Kitao, A., Hirata, H., Morita, A., Yoshida, T., Uchida, A., 1997. Transient damage to the axonal transport system without Wallerian degeneration by acute nerve compression. Exp. Neurol. 147, 248 – 255. Lubinska, L., 1958. ‘Intercalated’ internodes in nerve fibres. Nature (London) 181, 957 – 958. Lubinska, L., 1961. Demyelination and remyelination in the proximal parts of regenerating nerve fibers. J. Comp. Neurol. 117, 275 – 289. Ludwin, S.K., Maitland, M., 1984. Long-term remyelination fails to reconstitute normal thickness of central myelin sheaths. J. Neurol. Sci. 64, 193 – 198. Mason, J.L., Langaman, C., Morell, P., Suzuki, K., Matsushima, G.K., 2001. Episodic demyelination and subsequent remyelination within the murine central nervous system; changes in axonal calibre. Neuropathol. Appl. Neurobiol. 27, 50 – 58. Maxwell, W.L., Povlishock, J.T., Graham, D.L., 1997. A mechanistic
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analysis of nondisruptive axonal injury: a review. J. Neurotrauma 14, 419 – 440. Ochoa, J., Fowler, T.J., Gilliatt, R.W., 1972. Anatomical changes in peripheral nerves compressed by a pneumatic tourniquet. J. Anat. 113, 433 – 455. O’Leary, M.T., Blakemore, W.F., 1997. Use of a rat Y chromosome probe to determine the long-term survival of glial cells transplanted into areas of cns demyelination. J. Neurocytol. 26, 191 – 206. Prineas, J.W., Connell, F., 1978. The fine structure of chronically active multiple sclerosis plaques. Neurology 28, 68 – 75. Ransom, B.R., Stys, P.K., Waxman, S.G., 1990. The pathophysiology of anoxic injury in central nervous system white matter. Stroke 21 (Suppl III), 52 – 57. Reynolds, E.S., 1963. The use of lead citrate at high pH as an electronopaque stain in electron microscopy. J. Cell Biol. 17, 208 – 212. Ritchie, J.M., Rogart, R.B., 1977. The density of sodium channels in mammalian myelinated nerve fibres and the nature of the axonal membrane under the myelin sheath. Proc. Natl. Acad. Sci. U. S. A. 74, 211 – 215. Sanders, F.K., 1948. The thickness of the myelin sheaths of normal and regenerating peripheral nerve fibres. Proc. R. Soc. 135B, 323 – 357. Smith, K.J., Hall, S.M., 1980. Nerve conduction during peripheral demyelination and remyelination. J. Neurol. Sci. 48, 201 – 219. Smith, K.J., Blakemore, W.F., McDonald, W.I., 1981. The restoration of conduction by central remyelination. Brain 104, 383 – 404. Srinivasan, Y., Elmer, L., Davis, J., Bennett, V., Angelides, K., 1988. Ankyrin and spectrin associated with voltage-dependent sodium channels in brain. Nature (London) 333, 177 – 181. Stichel, C.C., Mu¨ller, H.W., 1998. Experimental strategies to promote axonal regeneration after traumatic central nervous system injury. Prog. Neurobiol. 56, 119 – 148. Waxman, S.G., 1981. Clinicopathological correlations in multiple sclerosis and related diseases. In: Waxman, S.G., Ritchie, J.M. (Eds.), Demyelinating Disease: Basic and Clinical Electrophysiology. Raven Press, New York, pp. 169 – 182. Waxman, S.G., Swadlow, H.A., 1977. The conduction properties of axons in central white matter. Prog. Neurobiol. 8, 297 – 324. Waxman, S.G., Black, J.A., Stys, P.K., Ransom, B.R., 1992. Ultrastructural concomitants of anoxic injury and early post-anoxic recovery in rat optic nerve. Brain Res. 574, 105 – 119. Williams, R.W., Chalupa, L.M., 1983. An analysis of axon caliber within the optic nerve of the cat: evidence of size groupings and regional organization. J. Neurosci. 3, 1554 – 1564.
Remyelination and recovery of conduction in cat optic nerve after demyelination by pressure Lynne J. Cottee, a C. Daniel, b Wai Sim Loh, a B.M. Harrison, c and W. Burke a,b,* a
b
Department of Physiology, Institute for Biomedical Research, University of Sydney, Australia Department of Anatomy/Histology, Institute for Biomedical Research, University of Sydney, Australia c Department of Biological Sciences, University of Technology Sydney, Australia Received 20 April 2000; revised 11 June 2003; accepted 13 June 2003
Abstract Pressure has been applied to the optic nerve of cats sufficient to block conduction in the large (Y) nerve fibers. The pressure block produces a mixture of axotomy and demyelination. By means of implanted electrodes, recovery of conduction in these fibers was monitored. There is a short-term recovery starting about 2 weeks after block induction and finishing at about 4 weeks. A later recovery starts at about 6 – 7 weeks and finishes at about 10 – 11 weeks. The remyelination has been monitored in the electron microscope by measurement of the myelin thickness and axon diameter of the large fibers. The remyelination follows a time course similar to the late phase of conduction recovery. By reference to the work of others, we surmise that the early recovery of conduction is due to the reorganization of microtubules disorganized by the pressure. D 2003 Elsevier Inc. All rights reserved. Keywords: Demyelination; Remyelination; Conduction recovery; Microtubules; Optic nerve; Cat
Introduction Demyelination causes a block in impulse conduction because the demyelinated axon membrane lacks an adequate regenerative ionic mechanism (Ritchie and Rogart, 1977). At a later time, sodium channels develop (Foster et al., 1980) and conduction again becomes possible, albeit now at very low conduction velocity, either continuous or saltatory (Bostock and Sears, 1978). Overlapping this process in time and eventually superseding it is the process of remyelination, which leads to a restoration of normal conduction. The exact relationship between the recovery of normal conduction and the remyelination is not clear. There are comparatively few reports on the time course of remyelination or the time course of restoration of conduction and no quantitative study on the two time courses in the same tissue. These remarks apply as much to the central nervous system (CNS) as to the peripheral nervous system. Time courses of remyelination in the central nervous system have
* Corresponding author. Department of Physiology, University of Sydney, NSW 2006, Australia. Fax: +61-2-9351-2058. E-mail address: [email protected] (W. Burke). 0014-4886/03/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0014-4886(03)00310-8
been described in (or can be inferred from) several reports (Clifford-Jones et al., 1980; Gledhill and McDonald, 1977; Gledhill et al., 1973; Harrison and McDonald, 1977; Hildebrand et al., 1985; Ludwin and Maitland, 1984; O’Leary and Blakemore, 1997; Smith et al., 1981). Reports of time courses of recovery of conduction are fewer (Felts and Smith, 1992; Fowler et al., 1972; Smith and Hall, 1980; Smith et al., 1981). In only one of these reports is it possible to compare the two time courses on neurons in the CNS. The paper of Smith et al. (1981) describes recovery of conduction in the spinal cord of the cat following demyelination by lysophosphatidylcholine; the recovery of conduction is not graphed and the remyelination is described only qualitatively. There have also been descriptions of recovery of function (Blight, 1993; Gledhill et al., 1973; Jacobson et al., 1979). One of these papers (Gledhill et al., 1973) also describes recovery of myelination. In the present paper, we give quantitative descriptions of the time courses of remyelination and of recovery of conduction in the same tissue, the optic nerve of the cat. Demyelination can be produced by pressure, by chemicals and by disease. The time courses of recovery may be different depending on the demyelinating agent and on the
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persistence of the agent. The advantage of using pressure is that once pressure has been withdrawn, all subsequent effects are due only to the initial pressure and are not dependent on any continued action by the agent. Selective pressure has been applied to the optic nerve of the cat with the intention of blocking conduction in the large (Y) fibers. The main purpose of this procedure was to elucidate the function of the Y fibers by comparing the effects of stimulating the Y-blocked nerve with the effects of stimulating the normal nerve on binocular cells in the brain (reviewed in Burke et al., 1998). When pressure is applied to a nerve, conduction in individual fibers may be blocked in at least five ways: (i) by hypoxia, (ii) by depolarization caused by accumulation of potassium ions and possibly other metabolites and by entry of calcium into the axoplasm, (iii) by disruption of the cytoskeleton, (iv) by demyelination and (v) by axotomy. The first two causes are quickly reversible, within minutes or hours. Recovery from disruption of microtubules may take days (Kitao et al., 1997). Recovery from demyelination takes weeks (see papers cited above), while there is no recovery from axotomy in the CNS unless special measures are taken (reviewed in Stichel and Mu¨ller, 1998). In our experiments on cats, we have applied pressure of sufficient magnitude and duration to produce a mixture of demyelination and axotomy of the large (Y) fibers of the optic nerve. In the electron microscope, the axotomised (degenerating) fibers are readily distinguishable from the demyelinated (or remyelinating) fibers. Hence, we were able to measure axon diameter and myelin thickness in the latter fibers at various times after the pressure block. In the same cats, with implanted electrodes, we could stimulate and record from optic nerve or tract and therefore could monitor any recovery of conduction in the Y fibers. The two time courses (morphological and functional) are different and a reason for this will be suggested.
Methods Surgical operation Cats were anaesthetized with sodium pentobarbitone, 40 mg/kg ip and as needed to maintain surgical anaesthesia. All operations were conducted with sterile or aseptic precautions. Bipolar stainless steel electrodes were inserted into optic tract (Fig. 1A, position 3) and intracranial optic nerve (Fig. 1A, position 2) using the response to a bright flash as a guide to position. One optic nerve (usually the left) was exposed by a dorsal approach. Fine enamelled stainless steel wire electrodes, the ends sharpened and bent back 1 – 2 mm, were hooked into the connective tissue adjacent to the junction of the nerve and eyeball (position 1) and cemented in place with cyanoacrylate glue. Using a special cuff 2 –3 mm wide (see Burke et al., 1986 for a full description) positioned a few millimeters
from the eyeball (Fig. 1A), we applied pressure to the exposed optic nerve sufficient to block conduction in the large (Y) fibers. Impulse conduction through the pressurized region was monitored by stimulating at electrodes 2 or 3 while recording the response (the compound action potential) from electrode 1. We applied pressure for no more than 30 s, then waited 1– 2 min before reapplying pressure. By starting at a low pressure (100 kPa) and gradually increasing the pressure with each successive application, it was possible to reduce the t1 response (the response of the Y fibers; Fig. 1B) with little or no effect on the t2 response (the response of the X fibers; Fig. 1B). We aimed to reduce the t1 response to less than 10%, without reducing the t2 response below 90%. Pressures near 200 kPa were usually needed to achieve this. When this had been done, the pressure device was withdrawn, the electrodes soldered to a 9-pin plug that was then firmly attached to the skull by dental acrylic, and the skin sutured. Buprenorphine, a long-lasting anaesthetic, 0.03 mg im, was given then and at 12-h intervals as required. Subsequently, the optic tract or nerve was stimulated and recorded from via the plug. Further details are given in previous publications (Burke et al., 1986, 1987, 1992, 1998; Cottee et al., 1991). Monitoring of responses Following the operation, we monitored the responses, at first daily, later at 2- to 3-day intervals, using single shocks to the intracranial optic nerve or tract. Our usual stimuli were 3 mA 0.05 ms; such stimuli produce a good but not maximal response. They do not produce aversive behaviour in the unrestrained animal, indeed may not even be perceived. By stimulating at electrodes 2 or 3 and recording the compound action potential from electrode 1, we determined whether the pressure block was maintained or whether there was any recovery of response. We also recorded from electrode 2 in response to stimulation at position 3 (or vice-versa) and used this response (Fig. 1C) to determine whether some fibers had been axotomised and ceased to conduct into the optic tract (see Results for a full description). Histology The aim of the histological procedures was to determine the myelin thickness in axons of various diameters. The blocked optic nerves were examined histologically at various times after the initial operation. Six normal nerves, either from pressure-blocked cats or unoperated cats, were also examined. The cats were deeply anaesthetized with sodium pentobarbitone (80 mg/kg ip) and respired with carbogen (95% O2, 5% CO2). The descending aorta was clamped and 5 ml of 1% sodium nitrite solution containing 500 IU heparin was injected into the left ventricle. One minute later, the head was rapidly perfused with 300 ml of warm (37 –40jC) 0.9% NaCl solution at pH 7.4. This was
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Fig. 1. Pressure block of cat optic nerve. (A) Diagram of peripheral visual system, to show eyes, optic nerves, optic chiasm, optic tracts and lateral geniculate nuclei (LGN). Electrodes are placed in right optic tract (ROT; position 3), left intracranial optic nerve (LICON; position 2) and in the left extracranial optic nerve (LECON) near the eyeball (position 1). Pressure is applied in the region 5 – 8 mm from the eyeball (shaded). Scale approximate. (B) Response at position 1 to stimulation at position 3 before the block (dark trace); after the block, the t1 response is virtually absent, while the t2 response is slightly reduced and delayed (faint trace) (cat LB165). (C) Response at position 3 to stimulation at position 2 one day after the block (dark trace); 4 days after the block, the t1 response has decreased to about 10%, while the t2 response is unchanged (faint trace) (cat LB152). In (B) and (C), negativity is upwards; the amplitudes of all responses were measured as positive peak to negative peak; from 5 to 32 responses were averaged for each datum point. (D) Amplitude of the t1 response recorded as in (C) as a function of time after pressure block. Note the sharp decline between 100 and 200 h (cat LB40).
followed by about 3 l of 0.1 M phosphate buffer containing 4% paraformaldehyde, 0.5% glutaraldehyde and 0.54% dextrose, adjusted to pH 7.4, and perfused over about 30 min, initially at a pressure of about 20 kPa, later at 2 –5 kPa. The nerves were dissected out 1– 2 h later and placed in a fixative solution the same as the above except for the glutaraldehyde concentration that was increased to 1%.
After overnight fixation at 5jC, the nerves were cut transversely into pieces 0.4 mm long using a vibratome, further fixed in 1% osmium tetroxide and then embedded in epoxy resin (Durcupan ACM, Fluka). Cross-sections of the nerves were taken from the pressurized regions or, in the case of the normal nerves, from a corresponding part of the nerve. Complete cross-
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sections cut at 1-Am thickness and stained with 0.25% toluidine blue in 0.5% dimethylaminoethanol were viewed in the light microscope to check for adequate fixation. Sections across one diameter of the nerve were cut at 70– 80 nm, stained with uranyl acetate and lead citrate (Reynolds, 1963) and viewed in a Philips CM12 microscope. Images were taken across the entire diameter at magnifications of between 5600 and 8800 times. These images were saved directly onto the hard drive of a PowerMac for later analysis. It was not our intention to measure all axons in a section since we were not aiming to obtain a population distribution. Because the pressure-blocking operation affected primarily the axons with diameters more than 5 Am, we concentrated on these. We adopted the value of 5 Am as criterion for the lower limit of Y fiber diameter and we have assumed that the t1 response (see later) is the response of the Y fibers (see Burke et al., 1986 and Cottee et al., 1991 for a discussion of these points). However, for purposes of comparison, a wide sprinkling of smaller axons was measured. Any axonal profiles that were degenerating or grossly distorted were omitted. Myelin thickness was measured on a compact part of the myelin. The axon perimeter and myelin thickness were measured using the PrismView data analysis program (Analytical Vision Inc., Raleigh, NC, USA); this program also calculated axon area. For the magnifications used, the limit of resolution (one pixel) was 0.010 – 0.016 Am. Axon diameter was determined in two ways. First, the diameter was taken to be the perimeter divided by p; this assumes that the length of the perimeter is not changed by the blocking procedure and that the axon usually has a circular profile. The plane of section of the nerves was orthogonal to their long axes. Of course, individual fibers can run obliquely but no correction was made for such fibers. It has been shown that axotomised axons in the peripheral nervous system tend to retain their circumference while losing volume, becoming crenated (Gillespie and Stein, 1983); we assume the same is true for injured central axons. Axon diameter was also calculated as the diameter of a circle with the same area as that measured. This assumes that the area as measured is the same as in life; this method gives slightly smaller diameters than the first method. We determined axon diameter by both methods for most experiments. However, the results shown in all the figures are based on the first method. The results were qualitatively the same for both methods. For statistical comparisons between groups, we used the Mann – Whitney U test; to test the significance of differences between slopes of regression lines, we used a t test (Goldstein, 1964). We adopted P V 0.05 for statistical significance. All procedures were carried out in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and approved by the Animal Care Ethics Committee of the University of Sydney.
Fig. 2. Recovery of t1 response in pressure-blocked optic nerve. (A) Response recorded as in Fig. 1B at various times after the block. Note the gradual recovery over about 5 weeks (cat LB158). (B) Graph of the changes in the t1 and t2 responses in a pressure-blocked optic nerve recorded as in Fig. 1B over about 7 weeks. Note the slow decline in the t2 response but recovery in the t1 response (cat LB40).
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Fig. 3. Graphs of the change in the t1 response in five pressure-blocked optic nerves. Recording as in Fig. 2 and normalization both to 0% (the weakest response post-block) and to 100% (final steady level). Note that four nerves show a two-stage recovery whereas one shows only the late recovery. One response for cat LB152 at 166 days has been omitted.
Fig. 4. Recovery of response in pressure-blocked nerves. The continuous solid line is the mean of the separate graphs in Fig. 3, interpolating where appropriate; averages were taken only when at least three values were available. The individual points and dashed line were obtained from measurements made (by us) in Fig. 5 of the paper of Smith et al. (1981), normalizing both to 0% (minimum response) and to a steady level at 100%. (In the data of Smith et al., there appeared to be a recovery to about 50% of the pre-block level.) Note the general resemblance of the two graphs.
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Results Degeneration versus demyelination Fig. 1A shows a diagram of the optic nerves, optic chiasm and optic tracts in the cat. Pressure is applied to one nerve close to the eyeball (shaded region) while monitoring conduction through this region by stimulating chiasm or tract at electrodes 2 or 3 and recording from an electrode at 1. A conduction block of the t1 response (the response of the Y fibers) is considered satisfactory when this response drops out leaving only the t2 response (the response of the X fibers): compare the responses in Fig. 1B. At this time, the responses from electrode 2 to stimulation at
electrode 3 (or vice-versa) show both the t1 and t2 responses (Fig. 1C) because the axons running between these two electrodes have normal conduction properties. However, if any axons have been axotomised by the pressure, their axons in this region will eventually lose membrane potential and cease to conduct. For Y fibers, this happens fairly abruptly on about the fourth day and the t1 response decreases (compare the responses in Fig. 1C; see Burke et al., 1986 for a full description). Note that when electrode 2 is in the chiasm, the response includes the response of some fibers not subjected to pressure block, that is, those fibers travelling ipsilaterally from retina to tract. To monitor only fibers travelling through the block site, it is necessary to place electrode 2 in the intracranial optic nerve; the elec-
Fig. 5. Electron microscopical appearance of normal optic nerve axons and axons subjected to pressure block. (A) Large axon in normal optic nerve. (B) Completely demyelinated medium-sized axon 27 days after pressure block (cat LB61). (C) Demyelinated – partly remyelinated large axon 53 days after pressure block (cat LB40). (D) Demyelinated – partly remyelinated large axon 53 days after pressure block (cat LB40). Note the extremely attenuated cytoplasm and the crenated myelin sheath. Scale bar (2 Am) applies to all sections.
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trode is satisfactorily placed when it records no response to stimulation of the contralateral eye. Depending on the particular circumstances attending the application of pressure, there may be a complete or only partial loss of t1 response in the region caudal to the block site (Fig. 1C). Fig. 1D exemplifies a case of large but nontotal loss of t1 response. The electrophysiological finding of a loss of t1 response at about 4 days is indicative of axotomy, a conclusion supported by morphological evidence of degeneration (Burke et al., 1986). It seems to be impossible to obtain a 90 – 100% t1 conduction block in the optic nerve without some, often considerable, degeneration. The conduction block in the nerve is maintained for at least a week, and often for several weeks, but usually there is some recovery of the t1 response, the extent of which depends on the degree of loss at 4 days (Figs. 1C, D); the greater the loss at 4 days, the less chance of recovery later. Recovery of t1 response Fig. 2A shows compound action potentials recorded from the optic nerve at electrode 1 in response to stimulation of the optic tract via electrode 3 at various times after a pressure block in one cat (LB158). Note that the t1 response appears at its normal latency (e.g. trace d). It is possible that during the process of recovery, individual Y axons may discharge at longer latencies, perhaps during the t2 response,
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but such discharges would probably be very asynchronous and could not be reliably detected. In Fig. 2B is the graph of recovery of the t1 response in another cat (LB40). Recovery commenced at about 20 days and reached a peak about 8 days later; there then seemed to be a later phase of recovery at about 42– 48 days. At the same time, there was only a decrease in the t2 response, a common occurrence. Fig. 3 graphs the t1 recovery curves in five cats. In all cases the responses have been normalized, to 0% for the smallest post-block response and to 100% for the final roughly steady level. Although there is considerable variability in the time courses, four of the five graphs reach 100% in about 30 days. Fig. 4 is a graph of the mean of the graphs in Fig. 3 (solid line). This emphasizes the two-stage recovery. To the graph of Fig. 4, we have added measurements we made on records of recovery of responses in the spinal cord of a cat treated with lysophosphatidylcholine (Fig. 5 in Smith et al., 1981; the short-latency response measured peak-to-peak; dashed line). The similarity of the two graphs will be considered in the Discussion. Remyelination Fig. 5 shows electron micrographs of a large axon in a normal optic nerve (Fig. 5A) and in nerves at 27 days (Fig.
Fig. 6. Relation of myelin thickness to axon diameter. (A) Plot of myelin thickness versus axon diameter for six normal optic nerves (data combined: 2216 axons), with regression line (solid line). The dashed lines are the regression lines for the 0- to 3-Am and >5-Am diameter axons; note the differences in their slopes from that of the solid regression line. (B) Plot of the myelin thickness/axon diameter (MT/AD) ratio for groups of axons of different diameters, with regression line. Points plotted at the median position of the group; SEMs plotted but smaller than the symbols in most cases.
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5B) and 53 days (Figs. 5C, D) after the pressure block. There is complete demyelination in the medium-sized axon shown in Fig. 5B at 27 days. Complete demyelination has been seen in other axons of similar size at this interval; the large axons had presumably either retained or reacquired myelin. At 53 days (Fig. 5C), remyelination was under way. In some axons at 53 days (Fig. 5D), the axoplasm appeared almost featureless and yet the axons did not appear to be degenerating; they may represent axons from which the axoplasm has been extruded by the pressure but without injury to the plasma membrane. In the fiber of Fig. 5D, the myelin appears crenated. On the assumption that the recovery of conduction was mainly due to remyelination, we attempted to substantiate
this idea by measuring axon diameters and myelin thicknesses at various times after the pressure block. Of course, in contradistinction to the electrophysiological measurements, we could not make serial measurements in a single animal but only a single measurement at a particular interval. We did not attempt to distinguish between partly demyelinated and remyelinating fibers. For comparison, we made measurements on six normal optic nerves. The data from all six nerves have been combined in a single graph in Fig. 6A. A regression line (solid line) gives a reasonable fit to the data, in agreement with several previous reports on both optic nerve (Bishop et al., 1971; Friede et al., 1971; Williams and Chalupa, 1983) and other tissues (Ludwin and Maitland, 1984; O’Leary and
Fig. 7. Plots of myelin thickness versus axon diameter for six pressure-blocked nerves. Data obtained at the post-block intervals indicated. Note that the slopes of the regression lines for axons of 0- to 3-Am diameter remain similar to those in normal nerves, whereas for axons >5-Am diameter, the slopes are radically changed (9 days, cat LB39; 27 days, cat LB61; 53 days, cat LB40; 79 days, cat LB158; 97 days, cat LB57; 167 days, cat LB152).
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Blakemore, 1997). Since our primary interest was in axons >5-Am diameter, we have added the regression line (dashed) for these axons, and for comparison, the regression line (dashed) for axons 0- to 3-Am diameter. The 0- to 3-Am group was chosen for comparison because the pressure block may affect some fibers with axon diameters less than 5 Am, but probably none with diameters less than 3 Am. It is evident that the single regression line is only an approximation for the whole population. The relation between myelin thickness and axon diameter is commonly depicted by graphing axon diameter against fiber diameter. The slope of this graph is the ‘g’ ratio and, in our terms, equals AD / (AD + 2MT). For cat optic nerve, Williams and Chalupa (1983) reported a value of 0.8 for the g ratio, while from Fig. 6A, this ratio is found to be 0.82. The MT/AD ratio is a simpler way of expressing the relationship and is used throughout this paper. It may be noted, however, that the MT/AD ratio is not constant but decreases with increase in axon size (Fig. 6B). It follows that the g ratio is likewise not constant but increases in the larger fibers, as has been observed previously for peripheral nerve with light microscopy (e.g. Sanders, 1948), but is not usually seen with the electron microscope (reviewed in Waxman and Swadlow, 1977). Fig. 7 shows graphs for myelin thickness versus axon diameter for various times after a pressure block with separate regression lines for the 0- to 3-Am group and the >5-Am group. It seems that the 0- to 3-Am group is essentially unchanged from normal at most intervals (no data were
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collected at 79 days); however, in the >5-Am group, the regression line has a much reduced slope, even becoming negative at 27 days but then increasing at longer intervals. Although the change in slope of the regression line gives an approximate measure of the time course of remyelination, this is not a good measure for several reasons. The regression line does not necessarily go through the origin. Also, if sampling is not extensive and does not include an adequate selection of fibers, the regression line will be distorted. A better way is to determine the myelin thickness/axon diameter (MT/AD) ratio for individual fibers and to compare these values with the corresponding ratios for normal fibers. It is necessary to make the comparison with the appropriate group of normal axons because of the decrease in this ratio with increase of axon diameter (Fig. 6B). The MT/AD ratios for diameter subgroups >5 Am are plotted against time after pressure block in Fig. 8. Each datum point is the mean MT/AD ratio in the pressureblocked nerve for a particular axon diameter range (e.g. 5 –6 Am) expressed as a percentage of this ratio for axons of the same size in the normal nerve. All the curves show a similar time course. The decrease to a trough at 27 days is interpreted as evidence of progressive demyelination. However, remyelination can commence within 2 weeks of a lesion (Smith et al., 1981). Hence, during this period, both demyelination and remyelination are occurring. Recovery of myelin thickness to normal occurs at about 70 days and is followed by a period when the MT/AD ratio exceeds normal at 79 and 97 days. Only the 5- to 6-Am group (at both days)
Fig. 8. Recovery of myelin thickness – axon diameter relationship. For axons >5-Am diameter, graph of the MT/AD ratio for five subgroups expressed as percentages of this ratio in the corresponding normal groups against time after pressure block. The separate graphs for axons of different diameter follow a similar time course to one another. For the points at 79 and 97 days, only those for the 5- to 6-Am group are significantly above the normal level (100%).
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Fig. 9. Comparison of recovery of conduction with recovery of MT/AD ratio. Graph of the MT/AD ratio for axons >5-Am diameter versus time after pressure block (symbols and left scale). All points except that at 167 days are significantly different from the normal value ( P < 0.0001 except for 79 days where P < 0.005). For all points, the SEMs are smaller than the symbol; the SEM for the ratio for normal axons is similar. For comparison, the mean response recovery curve from Fig. 4 is reproduced here (continuous line and right scale). The MT/AD ratio may also be read as a percentage using the right scale.
is significantly above normal ( P < 0.005), but this statistical significance and the similar pattern of the other groups suggest that this is a genuine effect. At these times, there is either a hyper-myelination or a relative shrinkage of the axon. All the curves fall to normal myelin thickness by about 170 days. The time course of remyelination is made clearer by combining the data in Fig. 8 and plotting the means in Fig. 9; all points are significantly different from normal except that at 167 days. Also plotted in Fig. 9 is the mean response recovery curve reproduced from Fig. 4. The two recovery curves are similar from about 50 days but differ considerably before that. There is an early phase of functional recovery commencing at 1 –2 weeks and peaking at about 4 weeks. At the critical time of around 30 days, the two curves are significantly different ( P = 0.04: Mann –Whitney, two-tailed). If the result from cat LB152 is excluded (this cat did not show the early phase of recovery; cf. Fig. 3), the difference is even more significant ( P = 0.006).
Discussion Method of pressure block The cuff we used was up to 3 mm wide when inflated. It was shown by Gilliatt et al. (Fowler et al., 1972; Ochoa et al., 1972) that pressure with a pneumatic cuff tends to produce a movement of myelin away from the centre of the cuff towards the uncompressed part of the nerve result-
ing in an invagination of the myelin at several nodes. From their data, we can safely assume that all the nodes under our cuff (2 – 4) were affected. It appears that the myelin that is stretched may be separated from its Schwann cell (in Gilliatt’s experiments) and degenerate, resulting in a ‘paranodal’ demyelination. The Schwann cell divides, its daughter cell(s) then proceed to remyelinate the demyelinated paranodal region to form new short ‘intercalated’ internodes (Lubinska, 1958, 1961). If this process also occurs in central axons, it would result in a mixture of relatively normal myelin and myelin in various stages of demyelination/ remyelination. Our sections would be expected to sample in both regions. This might account for the large scatter of myelin thicknesses observed at all intervals after the block (see Fig. 7). Although this means the experiment is less than ideal, provided we are always sampling an adequate number of fibers, we should still be able to chart the time course of remyelination accurately. In the present samples, we did not encounter a single large fiber that was completely demyelinated. This may have been due to sampling at intervals when axons had not completely demyelinated (9 days) or had remyelinated to a significant extent (27 days onwards). It seems that the medium-sized fibers are differently affected by the pressure, many showing complete demyelination at 27 days (Fig. 5C). Recovery of conduction When considering the reasons for recovery of conduction in fibers pressure-blocked in the present experiments, we
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can dismiss re-oxygenation and dispersal of potassium and other metabolites because the block persists for at least 10 days and recovery from these causes should be a matter of hours. On the other hand, axotomy would lead to degeneration of the caudal segment of the axons and would rule out any recovery of conduction in those fibers. The two remaining recovery processes (restoration of the cytoskeleton and remyelination) are both candidates in our experiments. Can recovery of conduction be due to remyelination? If we compare the time courses of recovery of conduction and remyelination in our experiments, we see that they are similar from about 50 days but that the early parts of the time courses are very different (Fig. 9). First, at 9 days, there is almost complete block of conduction, yet the myelin thickness is about 83% of normal, suggesting that the cause of the block is not simply demyelination. Secondly, at about 30 days, in most cases there is complete recovery of conduction (see Fig. 3) whereas there is now a significant amount of demyelination. Subsequently, a partial conduction block is reestablished transiently despite some further remyelination at this time, again suggesting another process. It is interesting that a similar delay in recovery of conduction is seen in the results of Smith et al. (1981) from the cat spinal cord demyelinated by lysophosphatidylcholine (see Fig. 4). Against these rather slow changes in conduction and remyelination, it should be emphasized that the initial block of conduction was achieved within a few minutes. This is more likely to be due to movement of axoplasm and stretching of the myelin sheath [leading to invagination of the myelin (Ochoa et al., 1972), and at greater pressures, rupture of the plasma membrane] than to stripping of myelin that, as Gledhill et al. (1973) found, continues to occur over at least a week. There are at least two possible explanations for the early recovery of function. The first is that the conduction block is due to a detachment of the myelin loops at the paranodes without detachment or loss of myelin elsewhere. The axon – glia connection at the paranodes (the ‘transverse bands’) is considered to be labile (Waxman, 1981) yet is critical for conduction because it effectively separates the extracellular and periaxonal spaces (Hirano and Dembitzer, 1978), and its detachment causes a significant fall in transverse resistance at the internode (Funch and Faber, 1984) and also the exposure of potassium channels (Chiu and Ritchie, 1981), both events reducing the safety factor for transmission. By the same token, such a detachment might be quickly healed. A second possibility for the early recovery of function assumes that the pressure causes a disruption of the cytoskeleton of the axons, specifically the microtubules. It has been demonstrated that mechanical injury to axons can cause stretching of the plasma membrane leading to entry of calcium ions which, either alone or in association with calmodulin, cause disruption and loss of microtubules
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(reviewed in Maxwell et al., 1997). Recently, Hanke (2002) has shown that pressure applied to the optic nerve of rats causes disruption of heavy neurofilaments and failure of axonal transport both anterograde and retrograde, with restitution commencing at about 12 days. It has also been shown that pressure causing a disruption of microtubules can lead to a behavioural deficit (Kitao et al., 1997). Kitao et al. (1997) used pressures of 12.5 and 25 g/mm2 on rat tibial nerve; they demonstrated that the axons were not axotomised but that the microtubules were disrupted (more severely at the higher pressure) and that recovery, estimated behaviourally (recovery of normal gait), was complete at 3 days (lower pressure) and 21 days (higher pressure). These pressures are roughly equivalent to 125 and 250 kPa and so are similar to the pressures used in our experiments; we usually needed pressures of 180 –220 kPa to produce a block of conduction in the large (Y) optic fibers. It is therefore very probable that there was disruption of microtubules in our experiments. Of course, the simplest explanation for the experiments of Kitao et al. (1997) is that disruption of microtubules caused inadequate release of transmitter at the neuromuscular junction or at other synapses, with no need to suppose any alteration in nerve conduction. However, in a study of the effect of anoxia on nerve conduction by Waxman et al. (1992), it was suggested that in addition to the entry of calcium into the axoplasm (Ransom et al., 1990) and disruption of the cytoskeleton, the latter might also include effects on the proteins ankyrin and spectrin that are associated with the sodium channels (Srinivasan et al., 1988), and this might in turn contribute to the loss of conduction. This system recovers relatively quickly (Kitao et al., 1997). At the present time, it is not possible to decide between these two alternatives. Indeed, they are not strictly alternatives since they are not mutually exclusive. A variant of this explanation is that the pressure completely removes axoplasm from a length of fiber without damage to the plasma membrane, as has been shown can occur in the squid giant axon (Baker et al., 1962). If this happens, one would expect recovery to be fairly slow (see Fig. 5D). Late depression and recovery It is not clear why there is a decreased response between the early and late recoveries (Figs. 3, 4). One possibility is that there may be two phases of demyelination, the first, a paranodal detachment, discussed above, not clearly evident in the histology and rapidly reversed. The second phase of demyelination might be due to the loss of myelin by separation from the oligodendrocyte soma. An individual axon might exhibit only one phase of demyelination but the population of axons in one nerve could exhibit both phases. There seems to be a discrepancy between our results and those of Harrison and McDonald (1977). These authors
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applied pressure to the spinal cord of a cat for extended periods, up to 3 h. The weakness of the limbs resulting from this procedure had almost completely disappeared at 1 month (Gledhill et al., 1973), whereas remyelination was not complete even at 18 months (Harrison and McDonald, 1977). Possibly, the demyelination in the experiments of Harrison and McDonald (1977) was more extensive or more severe than in our experiments. It should be noted that our pressure block affects only (or mainly) the Y fibers which constitute less than 5% of the total population in the cat’s optic nerve. Thus, the environment of the remyelinating fibers in our experiments may have been much more favourable than in the experiments of Harrison and McDonald (1977). Similarly, Ludwin and Maitland (1984) found that remyelination after the use of cuprizone in mice failed markedly to reach normal myelin thickness levels even at 5 –6 months. Since this drug presumably affects all parts of an axon, full remyelination might take a considerable time. Hyper-myelination? The remyelinating fibers appear to undergo a period of hyper-myelination between 70 and 150 days (Fig. 8). This elevated MT/AD ratio does not seem to be artifactual because the pattern is repeated for each group of fibers (5 –6 Am, 6– 7 Am, etc.) and it remains statistically significant for the 5- to 6-Am group (Fig. 8). Both peripheral and central axons shrink when demyelinated. Prineas and Connell (1978) found that demyelinated axons in multiple sclerosis plaques had reduced axon diameters. Reduction in axon diameter has also been observed in demyelinated axons of the corpus callosum of mice on a diet containing cuprizone (Mason et al., 2001). If axons shrink on demyelination but recover their volume after about 100 days, this might account for the changes in the MT/AD ratio. We think this is unlikely because the elevated MT/AD ratio occurs when we calculate axon diameter from the measured perimeter by dividing by p, and because there is evidence that the perimeter does not change with injury (Gillespie and Stein, 1983; see also Fig. 5D). Thus, this method of calculation would be indifferent to changes in volume. It remains a possibility that the perimeter of an axon does not stay constant. It is interesting that Sanders (1948) also describes a hyper-myelination in the peroneal nerve of the rabbit following axotomy by crushing and observation in the light microscope. Both the proximal and distal segments show increase in myelin thickness, in spite of a decrease in axon diameter in the proximal stump and in the regenerating axons of the distal stump. The changes are greatest at 200 days after crushing and are reduced at 300 days. Sanders (1948) interpreted the increase in myelin thickness as due to a loss of tension within the axon. Although this idea would not find favour today, nevertheless, measurements of the periodicity of the myelin and the number of lamellae would clarify the phenomenon. Without further
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