Ultrastructural changes in peripheral nerves of the fingers of three vibration-exposed persons with Raynaud's phenomenon.

H. Ultrastructural changes in peripheral nerves of the fingers of three vibration-exposed persons with Raynaud's phenomenon. Scand J Work Environ Health 14(1988) 31-35. A finger biopsy was performed on three patients with vibration-induced white finger (VWF), and the specimens were examined by electron microscopy for peripheral nerve changes. A characteristic neuropathy with perineurial fibrosis wasrevealedwhichwasoften accompanied by a thickened perineurium with a lamellar structure resembling onion skin. This peculiar fibrosis consisted of elongated cytoplasmic projections of perineurial cells or fibroblasts and a greatly increased amount of collagen which occasion ally contained fibrous long-spacing collagen. In the endoneurium, a decrease in the number of nerve fibers and a marked increase in collagen with fibroblasts were noted. Myelinated axons became smaller, and this occurrence suggested incomplete regeneration. The pathological changes were presumably the result of the long-term clinical course of VWF.

Changes in the peripheral nerves of patients with vibration-induced white finger (VWF) have been suggested from clinical and pathophysiological examinations by Lukas (8) and Torii et al (14), although Walton (15) failed to find such changes in his autopsy study. Experimentally, however, Karpova (7) recognized Wallerian degeneration in the peripheral nerves of rabbits. In their pathological examination of skin specimens from 30 patients with VWF, Takeuchi et al (11) found a clear increase in the thickness of the perineurium caused by connective tissue and marked perineurial fibrosis and muscular hypertrophy of the arterial walls, the last finding having also been described earlier by Ashe et al (1).
The objective of the present study was to determine the characteristics of the changes found in the peripheral nerves of VWF patients. For this purpose, we used electron microscopy, which allowed us to examine the pathological findings of the nerves in detail.

Subjects and methods
Finger biopsies of three patients with VWF were performed with the approval of the individuals concerned. The first patient was a 55-year-old man who had been exposed to vibration from a rammer for 12 years. He had had numbness, minor pain, and finally Raynaud's phenomenon in four fingers (II-V) of both hands for the last three years. The skin biopsy was performed in the region of the middle phalanx of the third finger and the distal phalanx of the fourth finger of the left hand by means of a skin-cutting procedure.
The second patient, a man 60 years of age, had used a pneumatic hammer and pick for 13 years. Thereafter, he complained of numbness and pain in his hands and fingers. Raynaud's phenomenon appeared in four fingers (II-V) of his left hand, and the attacks continued for several years after he stopped using vibrating tools. The biopsy specimen was taken from the region of the distal phalanx of the fourth finger of the left hand by the same method as was used for the first patient.
The third patient was a 68-year-old man who had had 15 years' heavy exposure to chain saws. He started complaining of numbness and pain seven years after he first began using the saws. He had also experienced severe symptoms of Raynaud's phenomenon in all fingers of both hands. Muscle atrophy of the hand was observed at the time of the biopsy. A reduced induction velocity of the radial nerve was also found in his right hand. The biopsy was performed in the area of the middle phalanx of the third finger of the right hand and the distal phalanx of the fourth finger of the right hand with the same method used for patient 1.
The biopsy specimens of all three patients were fixed in 2.5 0,70 glutaraldehyde and buffered with 0.1 M sodium cacodylate and 2 % sucrose at 4°C for one night. They were then postfixed in 1 % osmium tetroxide for 1 h. Routine embedding in epoxy resin and cutting on an ultrotome were performed. Samples of I 11m were routinely stained with toluidine blue and examined by light microscopy. Thereafter, the marked, thinly cut samples were stained with lead and observed under an electron microscope (Hitachi 12A). As new co ntro l specimens from humans co uld not be obtained, the speci me ns fro m five per sons fro m a p revio us st udy (11) were used as re ference also in th is st udy .

Results
In the exam ination by light microscopy, perin eurial fibr osis and a predominant thi ckenin g o f th e perineurium were noted in the specimens from a ll th ree patients. The perin eurium itself form ed a lam ella r struct ur e resem bling onion skin. Th is stru ctur e was ofte n seen on the sur face of the tra nsverse sectio n of th e peripheral nerve (figure 1). Co llagen tissue was increase d in the elonga ted cytoplasm of the per ineurial cells. Myelinated nerve fibers were frequently decreased in nu mber , and small nerve fibers not usually observed und er normal conditions were found. Axons were also decreased in number and were sm aller in size, wh ereas th e myelin sheat h was red uced in th ickn ess. The remaining nerve fibers were loo sely distribut ed in the endoneurium, where the connective tissue was increased. Sim ilar findings were ob served for the structure o f the peripheral nerves in all three individuals . On the other hand, the reference mat erial did not show a th icken ing of the per ineurium in any case, although the prepared slides were examined again in connection with the present study.
The electron microscopy revealed th e aforementioned findings and structures even more distinctly. The thickened concentric lamellar layers of the perineurium were composed of elon gated cytoplasmi c pro jections o f perineurial cells and greatly increased am ounts o f co llage n (figu res 2 and 3). The perineur ial cells themselves were increased, and their cytopl asmic projections were also extremely elongated. Some of the projection s in the inn er layer of the perineurium protruded into the endoneurium (figures 2 and 4). The nuclei, intracytoplas mic vesicles, and mitochondria of these cells had remained wit hin almos t normal limit s during the prolonged co urse of development of the revealed changes. Among the per ineurial cells and projection s which had a basement membrane (basal lam ina ), the amount of collagen was greatly increased and contained fibroblasts without a ba sement membrane . The co llagen bundles sometimes conta ined the so-called long-spa cing coll agen describ ed by Luse (9) , and the y varied in size and length ( figure 3) .
Nerve fibe rs were decrea sed in number to a considerable extent in the endoneurium, which had become loo se and edema tous as a res ult (figures 2 a nd 5). Axon s and myelinated sheaths were also decr eased in diam eter and thi ckness and therefore suggested inco mplete regeneration of ner ve fibers follo wing damage. Th e sma ll size obs erved was usuall y not seen in the reference mat erial. In addition the endo neur ium was infilt rated with collagen cont ainin g fibro blasts (figure 5).
Axo ns in the myelinated sheaths were smaller and more slender (figure s 4 and 5). They were distributed irregularly and were loosely scattered (figures I, 2, and 5). The num ber of mitochondria var ied and was sometim es increased (figur e 6), bu t there were no speci fic changes in th e fine structures, except for the smaller size. Vesicles o f irreg ular size and fo rm occas iona lly occ urred in the submyelina ted ar ea and in the matrix o f the axon s (figures 2 and 5). No unu sual character ist ics or struc tural cha nges were fo und in the individua l neurofilaments and neur otubules.
Myelin shea t hs were freq uently changed in the thinner layer , and someti mes th e cleft appea red to occur on ce or , rarely, twice between layer s o f lamellar myelin (figures 2 a nd 5). There was an occas io nal separ ation of lam ellae near th e Schm idt-Lantermann cleft, as well as marked chang es in the cleft, with myelin bodi es of different sizes a nd form s in the matrix.
Schwarm cells frequently contained a smaller myelinat ed axo n that varied in size in indi vidual cases; this axon had possibly been regenerat ed a nd sometimes ap-3 Mitochond ria are inc reased in number. (bar = 111m) peared as extremely fine in the cytoplasmic matrix near the nucleus (figures 4 and 5). Mitochondria were often increased in number in the cytoplasm and sometimes their structure was elongated ( figure 6). The glycogen particles were apparently increased in number, and some of them were also increased in size, forming considerably larger particles of rosettes, described as 13 particles by Drochmans (2).
The unmyelinated fibers were also decreased in number and size and were dispersed ( figure 5). This finding suggests possible regeneration after injury. Extremely fine axons without myelin were sometimes observed in the cytoplasm near the nucleus of the Schwarm cells ( figure 2).
In the endoneurium, bundles of collagen varying in size were loosely scattered, and homogeneous fluid suggesting edema filled the space between the collagen bundles and nerve fibers (figures 2 and 5). The collagen bundles were also increased in number and were accompanied by newly formed fibroblasts (figure s 4 and 5). Fibrous long-spacing collagen rarely occurred in the endoneurium. Similar findings were also observed around the thickened perineurium.
The changes found in patients 2 and 3 proved to be particularly marked .

Discussion
It is well known that Raynaud's phenomenon in association with occupational exposure to vibration is closely related to intense contraction of the local arteries, in which medial muscular hypertrophy occurs with an increase in the thickness of the arterial walls (I, I I). On the other hand, the clinical signs and symptoms of VWF indicate the presence of polyneuropathy (8,14), and Karpova (7) also suggested its occurrence from experimental data on rabbits. In addition Takeuchi et al (II) took biopsy specimens from the fingers of 30 patients with VWF and found that the per ipheral nerves were always damaged and that severe changes were evident.
In the present study nerve changes similar to those observed previously were confirmed for all three patients in the light microscopic exam ination. The findings indicated a decrease in the number of myelinated nerve fiber s and a characteristic appearance of perineuriallamellar thickness which was not observed in the reference material (II).
Under the electron microscope the first notable feature was thick lamellar fibrosis of the perineurium which resembled onion skin in transverse sections . The thick perineurium consisted of elongated cytoplasmic projections of increased perineurial cells with a greatly increased amount of collagen among them . Such thick perineurial fibrosis has not been reported in other forms of polyneuropathy described in the literature (3,5,6,10,12,13) . The amount of collagen was also increased around the perineurium itself, and this finding also indicated perineurial fibrosis . Such a marked in-34 crease in collagen presumably resulted from the longterm presence of repeated vibration and th e separation of the nerve bundles from the surro unding tissue as a result of expo sure to intense vibration. A similar mechanism possibly operated around the artery and led to the thick periarterial fibrosis observed in one of our previous studies (II).
Another characteristic determined with the aid of electron microscopy was a decrease in the number of nerve fiber s. The ner ve fiber s were often extremely small in size, and the myelinated axons were also smaller, presumably suggesting some regeneration similar to that which occurs in the polyneuropathy related to Minamata disease (4). The Schwarm cells were increased in number and were often rich in mitochondria, which were probably active in the regeneration of myelinated nerve fibers with thinner myelin sheath s. Therefore, the Schwann cells often contained a smaller or fine axon within their cytoplasm. The decrease in the number of nerve fiber s resulted in an increa sed amount of collagen with fibroblasts in the endoneurium, and it also caused the perineurial cells to project into the endoneurium. The borderline areas between the perineurium and endoneurium therefore became unclear and broader and were infiltrated by fibroblasts . The long-term clinical course of the VWF presumably led to these pathological changes.
The aforementioned findings suggest that repeated exposure to intense vibration may produce a characteristic nerve disturbance which is somewhat different from toxic or metabolic neuropathies, although it remains to be confirmed whether other physical mechanisms may produce a similar fibrou s thickening of the perineurium .