98

JNS 03427

Journal of the Neurological Sciences, 100 (i 990) 9~-~¢)7 [:J~ex icr

A fluorescence study of changes in noradrenergic sympathetic fibres in experimental peripheral nerve neuromas

J e n n i f e r R u t h Smal l , J o h n Wi l l i am S c a d d i n g a n d D a v i d Ne i l L a n d o n

Department of Clinical Neurology, Institute of Neurology, Queen Square, London (U.K.)

(Received 3 April, 1989) (Revised, received 23 July, 1990)

(Accepted 23 July, 1990)

Key words: Sympathetic efferent activity; Noradrenaline; Pain; Experimental neuromas.

Summary

Experimental neuromas were produced in rats by sciatic nerve section and avulsion of the distal stumps. At intervals varying from 3 days to 8 weeks after nerve section, the developing neuromas were resected and processed for noradrenaline (NA) fluorescence microscopy by the sucrose - phosphate - glyoxylic acid (SPG) method. From serial longitudinal sections through the neuromas and the nerve proximally, counts of noradrenergic sympathetic axons were made, together with qualitative observations of axon sprouting and NA content. By 3 days after nerve section there was a massive sprouting of sympathetic axons, with increased NA content, particularly towards the distal tip of the neuroma. Axon counts remained high 1 week following section then fell to below normal levels at 2 weeks, returning towards normal 8 weeks after nerve section. These results are discussed in relation to the known pathophysiological interaction between sympathetic efferent and sensory afferent fibres, which develops in neuromas following nerve section.

Introduction

There is substantial clinical evidence that noradrenergic sympathetic efferent activity is important in maintaining pain which may be associated with some chronic peripheral nerve lesions (Bonica, 1979; Loh and Nathan, 1978). Post- traumatic neuralgia, including causalgia, may follow a wide variety of nerve lesions, and may be relieved to some extent by sympathetic blockade in at least 50~ o of cases (Betcher et al., 1953; Lob and Nathan, 1978; Scadding, 1982). The important conclusion from these clinical observations is that noradrenergic sympathetic efferent activity has a major influence on the maintenance of pain associated with peripheral nerve lesions in many patients.

Physical injury to a nerve, such as crush or section, results in an acute, transient injury discharge (Adrian, 1930; Wall et al. 1974). Following this initial barrage, a chronic ongoing sensory afferent barrage develops (Wall and Gutnick, 1974; Govrin-Lippmann and Devor, 1978), which is associated with the regeneration of axons. Regenerating sensory fibres are also highly mechanically sensitive (Wall and Gutnick, 1974; Scadding, 1981 ; Blumberg and J anig, 1984) and show a specific sensitivity to NA. This adrenergic

Correspondence to: Dr. J.R. Small, Department of Clinical Neurology, Institute of Neurology, Queen Square, London WC1N 3BG, U.K.

sensitivity may be demonstrated either by intravenous administration of NA or adrenaline (Wall and Gutnick, 1974; Korenmann and Devor, 1981 ; Scadding, 1981), or by the enhancement of the afferent impulse barrage from neuromas by selective orthodromic electrical stimulation of sympathetic efferent fibres which end in the neuromas, via the lumbar sympathetic trunk (Devor and Janig, 1981; Blumberg and Janig, 1984). The latency of this response to lumbar sympathetic trunk stimulation is long and variable. The development of synapses has been proposed to account for this interaction between sympathetic efferent and sensory afferent fibres, but light and electron micro- scope studies have not revealed such specialized forms of contact between fibres (Cajal, 1928; Spencer, 1971 ; Morris et al., 1972; Scadding, 1982). However, the long and vari- able latency favours a looser form of interaction (Devor and Janig, 1981; Korenmann and Devor, 1981; Scadding, 1982) in which NA, present in the sympathetic fibres of the neuroma, stimulates sprouting afferent fibres, presumably via the development of alpha-receptors on the axolemmae.

In injured peripheral nerves, NA, identified as fluo- rescent material, accumulates proximal to a crush or liga- tion (Dahlstrom and Fuxe, 1964; Kapeller and Mayor, 1967). This accumulation proceeds at a linear rate for the first 48 h following surgery (Dahlstrom and Haggendal 1966), but from 48 to 96 h after surgery, the rate of accumu-

0022-510X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

lation ceases to increase due to the cell body reaction, which results in depression of the rate of synthesis and transport of the storage granules (Grafstein, 1975). Following this reaction, the amount of NA in the nerve returns towards normal, but the time taken for NA levels to return to normal depends on the type of injury sustained, and cutting or ligating the nerves causes a severe and more permanent depletion than a crush (Karlstrom and Dahlstrom, 1973). Studies using retrograde HRP labelling of sympathetic ganglion cells proximal to a mixed peripheral nerve neuroma suggest that about two-thirds of sympathetic efferent fibres ending in the neuroma eventually degenerate (Janig and McLachlan, 1984). In contrast, an investigation of the concentrations of noradrenaline in experimental neuromas by bioassay has shown a marked initial rise during the first week after nerve section, followed by a fall to about 50~o of the normal value by two weeks, with recovery to normal levels eight weeks after nerve section (Scadding, Brown and Cauthen, unpublished results).

The present paper reports the results of a morphometric study of the changes in sympathetic axon numbers in experimental neuromas. It was undertaken as part of a larger investigation of the pathological interaction which develops between noradrenergic sympathetic efferents and sensory afferents in regions of peripheral nerve damage, because of the importance of this interaction in relation to pain.

Materials and methods

Experiments were conducted on 21adult female Wistar rats, weighing between 170 and 200 g. The animals were anaesthetized with a mixture of fentanyl (0.1575 mg .kg-1 ) , fluanisone (5 m g . k g - l ) and midazolam (2.5 mg. kg - 1 ), in 2 ml. kg - 1 of water for injection. With sterile precautions, the left sciatic nerve was exposed in the pop- liteal fossa and lower thigh, and dissected free from surrounding tissues. The nerve was then sectioned in the lower thigh with a pair of fine scissors, and the distal stumps of tibial, peroneal and sural nerves avulsed, to permit neuroma formation. The skin was closed with silk sutures, and the wound sealed with Nobecutane plastic spray.

Fluorescence microscopy At 3 days and 1, 2, 3, 4, 6 and 8 weeks following nerve

section, the animals were reanaesthetized and the left sciatic nerve exposed. The whole of the neuroma together with 5 mm of proximal nerve attached was excised. In each animal a similar length of the normal right sciatic nerve was removed at the same level, as a control. Both nerves were orientated for longitudinal sectioning, with the proximal end

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towards the top of the block, embedded in fresh liver for mechanical support, mounted on cork chucks, and plunged into isopentane cooled by liquid nitrogen. Six further con- trol nerves were removed from three normal unoperated rats, and treated in a similar fashion. 10-#m frozen sections of the nerve were cut in a cryostat and every fourth section treated with glyoxylic acid, according to the SPG fluo- rescence method of De la Torre and Surgeon (1976), for the demonstration of monoamines: sections were collected singly onto glass slides and dipped after a few seconds air drying into a freshly made solution of 1 ~o glyoxylic acid in 0.2 M sucrose and 0.23 M potassium phosphate buffer at pH 7.4 (SPG solution) for 3 sec, after which time slides were dried in a cool air stream for 10-15 min. The fluo- rescence reaction was developed by heating the slides in an oven at 80 ° C for 5 min. Slides were then coverslipped using liquid paraffin, and the sections examined in a Nikon Labophot fluorescence microscope equipped with an epi- fluorescence system, BV-2A excitation and Blue barrier (470 nm) filters.

The numbers of green/blue fluorescing noradrenergic axons across the width of each section were counted at 5 different sites: 0.5, 1.0, 1.5, 2.0 and 2.5 mm proximal to the point of nerve section. The remainder of the more proximal part of the section was assessed qualitatively. Distances were measured using an eyepiece graticule cursor and the Vernier scale on the stage of the microscope: the eyepiece graticule cursor was orientated such that it crossed the section perpendicular to its longitudinal axis, and fluorescent axons which were bisected by the cursor were scored positively (Fig. 1). 20 sections from each nerve were counted, thus 1.6 mm of the thickness of each nerve was sampled. The counts for the 20 sections were then pooled for each distance, and the mean number of sympathetic axons at each of the 5 distances calculated.

Reserpine Following left sciatic nerve section, two rats were treated

with reserpine, at a dose of 10 mg. kg- ~ dissolved in 0.1 M acetic acid and given intraperitoneally 24 h before biopsy at 3 days after nerve section. The right and left sciatic nerves were removed, treated for fluorescence microscopy as described above, and assessed qualitatively.

Statistical analysis of the mean number of fluorescent axons in the neuromas was performed with the use of t-tests.

Results

Normal nerves Fluorescent axons were present throughout the normal

sciatic nerve examined, evenly distributed along the length of each nerve. No significant variation was found in the

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T A B L E 1

C O N T R A L A T E R A L N O R M A L N E R V E S

M e a n number of f luorescent axons

Dis tance from nerve tip: 0.5 mm 1.0 m m 1.5 m m 2.0 m m 2.5 mm

n = 2 1

Unopera t ed normal nerves

n = 6

187" 220 216 210 201

-+11 _+13 _+11 _+11 -+10

190 202 194 199 186

+ 17 _+21 +21 _+ 19 _+21

Neuromas

Dis t ance from dis ta l tip: 0.5 mm 1.0 m m 1.5 m m 2.0 m m 2.5 m m

3 day neu roma n = 2 - 717 604 554 484

_+96 +91 +_31 _+ 1

7 day neu roma n = 3 855 609 496 397 349

+ 125 -+ 67 -+ 36 -+ 35 + 17

14 day neu roma n = 5 111 154 161 165 171

+ 10 -+ 17 ± 18 _+ 25 ± 24

21 day neu roma n = 4 310 261 236 203 184

+ 15 _+22 _+32 -+20 _+ 15

28 day neu roma n = 3 351 264 230 197 183

+ 07 +_ 22 + 14 -+ 20 + 21

42 day neu roma n = 3 273 221 186 162 141

+ 3 4 ±11 + 0 6 +15 ± 9

56 day neu roma n = 3 235 210 198 177 171

+13 _+8 +_11 _+8 + 9

* Number s represent m e a n counts from 20 sect ions _+ SE.

numbers of fluorescent axons along the course of the indi- vidual nerves (Table 1, Fig. 2). Each fluorescent axon appeared bright and uniform in diameter along its length, and contained varicosities, although individual axons varied in calibre. Axons were irregularly distributed across the nerve and sometimes seen in groups. The outlines of the myelinated fibres were visible, but did not fluoresce (Fig. 1). Sample counts of sympathetic axons in normal sciatic nerves from non-operated animals were not significantly different from the normal sciatic nerves contralateral to neuromas in operated animals (Table 1), thus there was no effect on the sympathetic axon population of the right sciatic nerve when the left sciatic nerve was sectioned. The right nerves contralateral to neuromas were thus suitable controls, and the statistical analysis was restricted to the use of these nerves as normal controls.

3-day neuromas Very large accumulations of granular fluorescent

material were seen at the distal tip of the neuroma, making

identification of individual axons 0.5 mm proximal to the neuroma tip difficult (Fig. 4), and therefore no attempt was made to quantify the fluorescence in these neuromas at this point. Individual axons could be resolved under the micro- scope 1.0 mm from the tip, and therefore counts were begun here. Counts made 1.0 mm from the tip of the proximal stump showed that fluorescent axon numbers were signifi- cantly increased to over 3 times the normal value, (Table 1, P < 0.001), and individual axons were swollen. Estimates based on counts from 1 week neuromas (Table 1) suggest that the numbers of fluorescent axons may rise to 5 x normal levels at the distal tip of three day neuromas (Fig. 2). The accumulation of fluorescence persisted throughout the length of the nerve observed, so that at a distance of 2.5 mm from the tip, counts were still 2.5 x normal (Table 1, Fig. 3, P < 0.001). Sprouting was in evidence at 3 days; a few very fine beaded axons were seen growing from the edge of the distal tip of the neuroma. These sprouts were orientated either parallel to other fibres or at right angles to the main body of the nerve.

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Fig. 1. Longitudinal section of normal sciatic nerve, showing the distribution of fluorescent sympathetic axons, and the method used for quantifying fluorescence. The position of the cursor is shown, and the count of fluorescent axons at this point = 7; x 80.

--- 1000 C

E

" " 800

0 X m

E soo

U

0

400

200 E

z

a

\

e.- - - - - ' ' - - ¢ _; , ,~_ ~=, " .

y mean 3 day

y mean 7 day

y mean 14 day

y mean normal

! !

0 1 2 3

Dis tance (mm)

Fig. 2. Sympathetic axon counts from normal and transected rat sciatic nerves, from 3 to 14 days following section, showing the variation in counts along the nerve in a distal - proximal direction. Normal nerve shown at time 0 days.

1000 la, %

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102

' I ' I I ' ' I ' I ' i " l

3 7 14 21 28 42 5G

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Time (days) Fig. 3. Sympathetic axon counts from normal and transected rat sciatic nerves, from 3 to 56 days following section, 0.5 and 2.5 mm from the distal

tip

Fig. 4. Distal tip of a neuroma 3 days after sciatic nerve section, showing a massive accumulation of noradrenaline, and lack of resolution of individual axons. This area was not assessed quantitatively. × 40.

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1-week neuromas

One week after nerve section the numbers of fluorescent axons were still greatly increased, by 4.6 x at 0.5 mm from the distal tip (Table 1, P < 0.001), and remained high at all points measured proximally, being approximately twice normal 2.5 mm from the tip of the neuroma (Table 1 and Fig. 2, P < 0.001). Many axons were still very swollen, but there was a large network of very fine intermingling sprouts at the neuroma tip, and some sprouts were again observed running at right angles to the main body of the nerve (Fig. 5). A few axons contained large collections of fluo- rescence along their length, larger than normal varicosities, and of different shape, most prominently at a distance of 1 mm from the tip of the neuroma (Fig. 6).

2-week neuromas

Two weeks after nerve section the numbers of fluorescent axons had decreased to 60~ of normal at 0.5 mm from the distal tip (Table 1, Fig. 2, P < 0.001). There were no large collections of fluorescence, as seen earlier, and axons at the tip of the neuroma were thinner than normal, with intermingling sprouts in the new growth distal to the estimated point of nerve section. Fluorescent

axons were more numerous further proximally and of greater calibre. At 2.5 mm proximally, total fluorescent axon counts were still reduced, at 90~o of normal numbers, but this difference was not statistically significant (Table 1, Fig. 2, P > 0.1). A few yellow autofluorescent mast cells were visible, but only towards the distal tip.

3 - 4 week neuromas

Three and four weeks after nerve section there were increased counts of axons at the distal tip, so that 4 weeks after nerve section, counts were nearly twice normal (Table 1, Fig. 3, P < 0.001) but were not significantly dif- ferent from normal at 2.5 mm proximal to the estimated point of nerve section, (Table 1, Fig. 3, P > 0.5). In the distal part of the neuroma there were many intermingled fluorescent axon sprouts, some with abnormally large vari- cosities, extending more than 2 mm distal to the estimated original point of nerve section. Some axons were still sprouting in a direction perpendicular to the main nerve (Fig. 7). Blood vessels were prominent at this interval after nerve section, but their sympathetic innervation was dis- tinguishable from that in the nerve trunk proper on the basis that it was somewhat coarser (Fig. 8).

Fig. 5. Distal tip of a neuroma 7 days after nerve section with an extensive network of sympathetic axonal sprouts (arrows). x 80.

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Fig. 8. New tissue distal to the original section point, showing distinctive vascular innervation in a neuroma 4 weeks after section (BV = blood vessel). × 80.

6-8 week neuromas

Six and eight weeks after nerve section counts at the distal tip were lower than at earlier times after nerve section (Table i). The numbers of sprouts appeared to be fewer, although there were still significantly higher counts at the distal tip when compared to the more proximal portion of the nerve (Fig. 3). There was a smaller significant difference between counts at 6 weeks at the 0.5 mm level and normal counts (0.02 > P > 0.01), compared to the 4 week neuro- mas. Fluorescent axons were thinner than in the normal nerves, especially at the tip, and larger collections of yellow autofluorescent mast cells extending for 1-2 mm proximally were seen, accompanied by a much increased vascularity in the distal tip and surrounding perineurium.

Reserpine-treated animals

Three days after nerve section there was a marked reduc- tion in numbers of fluorescing axons, particularly when compared with 3-day neuromas in rats not treated with reserpine. Very small patches of diffuse fluorescence were seen at the distal tip, but there were virtually no axons fluorescing in the remainder of the nerve.

Discussion

In this study, fluorescent material, demonstrated by the SPG method, was seen to accumulate in axons of develop- ing sciatic nerve neuromas. The SPG method is specific for monoamines (De la Torre and Surgeon, 1976) and virtually complete depletion of fluorescence by reserpine supports our conclusion that the fluorescence observed in our experi- mental material resulted from the presence of NA. After interruption of axons by crush, ligation or section, tissue levels of noradrenaline rise due to a block in the proximo- distal axonal transport beyond the point of injury, and an initial rise in the rate of transport of amine granules to the proximal stump (Dahlstrom and Haggendal, 1970). In our study the maximum accumulation of fluorescence occurred at three days, although the timing of the absolute peak could not be determined with precision as nerves were only sampled at three and seven days during the first week after nerve section. It is however probable that by 3 days (72 h) following section the accumulation rate has ceased to increase and is in the plateau phase reported by Karlstrom and Dahlstrom (1973) and Ohshiro et al. (1978).

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The cessation of noradrenaline accumulation after 48 h is due to the reaction of the cell bodies to injury, resulting in a decrease in synthesis and transport of amine granules, rather than to a change in monoamine oxidase levels (Boyle and Gillespie, 1970). In addition, the transmitter is mem- brane bound in granules and is therefore not available for

metabolism (Dahlstrom and Haggendal, 1970). The cell bodies of damaged neurones show a net decrease in their neuronal content of certain proteins, including the mono- amine oxidases and the synthetic enzymes dopamine beta- hydroxylase and tyrosine hydroxylase, due to increased proteolysis (Lieberman, 1971; Grafstein, 1975). There is also a change in the pattern of protein synthesis following axotomy away from transmitter synthetic proteins in favour of structural components, which may contribute to a fall in noradrenaline synthesis (Griffith and Lavelle, 1971).

The large numbers of fluorescent axons seen at three days following nerve section may result not only from accumulation of noradrenaline in axons which might other- wise not reach the level of visibility, but also from an increase in the actual numbers of axons present due to sprouting. In keeping with our observations, McQuarrie et al. (1978) reported a 75~o increase in sympathetic axon counts in rat sciatic nerve at 2 weeks after tibial nerve transection and 3 days following subsequent sciatic nerve crush.

By 7 days following section, the number of fluorescing axons was much reduced, although still well above normal levels. Counts of adrenergic axons fell rapidly to a minimum at 2 weeks, which was below normal 0.5 mm from the distal tip.

Four weeks following injury, counts of fluorescing axons had risen significantly, particularly at the distal tip of the neuroma. This increase was probably due to increased visibility of sprouting because of an increase in NA content of axons following a rise in the rates of transport and/or synthesis. Eight weeks following nerve section the number of sympathetic axons in the normal nerves and neuromas did not differ significantly. In an ultrastructural study of myelinated and unmyelinated axon numbers in sectioned and ligated rat sciatic nerves, Jenq and Coggeshall (1985) found that the number of unmyelinated axons in the proximal end of a sectioned nerve which was reapposed to the distal stump in a tube at the region of transection, 8 weeks following injury, was close to that in a normal nerve. Akhough unmyelinated afferent C fibres must also have been counted, this study closely parallels our findings.

Our results show that the NA content and number of sympathetic fibres in neuromas vary considerably with time following nerve section. Physiological experiments have demonstrated that ectopic impulse generation in neuromas can be enhanced by adrenaline and noradrenaline (Wall and Gutnick 1974; Scadding 1981 ; Korenmann and Devor,

1981). A quantitative study of ongoing activity, mechano- sensitivity and adrenaline sensitivity in mouse sciatic nerve neuromas revealed an early peak of these abnormal activ- ities at 3-4 days, to be followed by a later and larger peak at 14-21 days (Scadding, 1981). In this study the sampled activity was predominantly in myelinated fibres, but similar properties including NA sensitivity have been observed in axotomised C fibres forming a neuroma (Habler et al. 1987). The relationship between the abnormal afferent impulse barrage arising in neuromas to painful sensation remains controversial. In man, ectopic impulse generation in areas of nerve damage has been demonstrated by microneuro- graphy (Ochoa et al., 1982; Nordin et al., 1984), and such activity correlates with the subjective experience of painful paraesthesiae. There are no direct observations by micro- neurography in man which allow conclusions to be drawn concerning the role of endogenously released catechol- amines in maintaining or enhancing ectopic impulse gen- eration in areas of nerve damage such as neuromas, but the clinical evidence discussed in the introduction indicates that this property is of considerable importance in many patients. The beneficial effect of regional sympathetic blockade suggests that locally released rather than cir- culating catecholamines are important in producing and maintaining pain in nerve injury in many patients, and this conclusion is supported by the experimental observations of Devor and Janig (1981).

The results of our study do not shed light on the nature of the abnormal interaction which develops between sympathetic efferent fibres and sprouting sensory afferent fibres in neuromas, but they do show that large amounts of noradrenaline are available within the region of nerve dam- age, and that sympathetic efferent fibres are still sprouting several weeks after nerve section within the neuroma, which is the site of the pathophysiological interaction. Electron microscope studies are currently in progress to elucidate further the anatomical relationship of sympathetic and other axons in neuromas, and the effects of drugs which may have therapeutic importance in limiting these inter- actions are also under study using this model.

Acknowledgements This study was supported by a project grant from the MRC. Preliminary results have been presented at the 7th Meeting of the British Neuropathological Society, London, U.K., January 1989.

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