Intraneural edema following exposure to vibration

1. Intra neural edema following exposure to vibration. Scand J Work Environ Health 13(1987) 326-329. Periph eral neuropathy represents a well-knowncomplication from long-term exposure to vibration. In the pres ent study an experimental model is presented with the purpose of analyzing the formation of intraneural edema following vibration exposure. Vibration (82 Hz, peak-to-peak amplitude 0.21 mm) was induced in the hind limb of rats by the use of vibrating electric motors during 4 hid for 5 d. Tracer techniques (with albumin Evans blue and horseradish peroxidase) were used to study the permeability of intraneural microvessels after the vibration exposure on day 5. It was found that the vibration trauma in this model induced epineurial edema in the sciatic nerve. It is hypothesized that the formation of intraneural edema may be an important pathophysiological factor in the occurrence of vibration-induced neuropathy.

" W hite fingers" (Raynaud' s phenomenon) and peripheral neuropathy rep resent well-know n co m plicatio ns from long-t erm expos ur e to vibra tio n . Although the peripheral vasc ula r pr oblem has gained mu ch attention (I, 2, 17), the neurological disturbances ha ve no t been so well recognized. However, ov er recen t yea rs numbness, paresthesia, and muscle wa sting have been observed , often in association with impaired cutaneous vibrotactile sensibility (5), decreased nerve conduction veloci ty (14) , and decrea sed mu scle power of the hand gr ip (2,3,4) .
In th e upper extremity the neuropathy usually invol ves the median or ulnar ner ves, more seld o m both at one and the same time (7) . In early stages the symptoms may im itate those seen in carpal tunnel syndro me although conduction velocity across the ca rpal tunnel is so meti mes normal. The pathoanatomic ba sis for these lesions is not known, although some authors claim th at they have ob served degenerative fiber changes in nerves subject ed to vibration (6) and in biopsies from fingers o f vibratio n -expose d workers (16) .
Peripher al nerves a re well vas cu larized st ru ctures , responding to trauma with a n in fla m ma to ry rea ction including increased vascular permeability and th e formation of intraneural edem a (8,9,10). A n intraneural edema m ay produce serio us acute cons equences for normal nerve fiber function by changing th e endoneu-rial co m pos itio n o f electrolytes a nd increa sing endoneurial fluid pr essure (9,II ,13). C hr o nic ede ma m ay be invaded by fib ro blasts and transfo rmed to a n intraneur al scar. There is reason to believe that th e early stages of ca rpal tunnel synd ro me , cha rac terized by reversible no cturnal paresthesias in the hand, are based upon edema formation and vas cular insufficien cy occur ring in the nerve during the night (10,15).
The purpose of the present investigation was to study the effects of vibration on intraneural microvascular fun ction with special reference to the fo rma tion o f intrane ura l ede ma. Our hypoth esis was that intraneural ede ma migh t be an etiol og ic facto r in the o ccu rr en ce o f vibra tio n-in du ced neuropathy.

Materials and methods
The study was carried out on Sprague Dawley rats (body weight 200-250 g), anesthetized by intraperitoneal injections of a solution composed of sodium pentobarbital (60 mg/ml) , 0.9 % saline, and diazepam (5 mg/ ml) in I: 1:4 volume proportions. Vibration of controlled frequency and intensity was induced in one of the hind limbs of the rats during 4 h/ d for 5 d. After the vibration exposure and perfusion by a tracer (see the following text) on the fifth day, the animals were killed, and the sciatic nerves were removed.

The vibration exciter
A simple method was developed for inducing vibration in the hind limb of the rats. Small electric motors were modified so that a counterweight of defined mass was fixed to the axis in an eccentric position. Increasing the voltage of the motor resulted in vibration of increasing frequency. At 14 V the motor induced vibration of 82 Hz and a peak-to-peak amplitude of 0.21 mm in the system.
The motor was enclosed in a silicone block. To compensate for the temperature increase in the system, air of room temperatur e was passed through two channels in the silicone mass close to the motor. A slit fitting the shape of the foot of the rats was made in the silicone block. The rats were sus-pended in a cradle with one hind foot placed in the slit. In this manner, the hind foot and limb were subjected to vibration of defined frequenc y, amplitude, and dur ation.

Meth ods f or assessing vascular permeability
Evans blue-albumin. Serum albumin, labeled with Evans blue, has been used previously in numerous studies for assessing micro vascular permeability in periph eral nerves (8,9). A solution , here called EBA, was prepared by mixing 5 0" /0 bovine albumin with Evans blue. After the vibration exposure on da y 5, EBA (I ml per 100 g of body weight) was slowly injected into a tail vein or via a catheter in the femor al vein of the limb not subjected to vibration and allowed to circulate for 30 min . A segment of the sciatic nerve from the vibrated leg was then removed and fixed in 4 070 buffered formaldehyde solution for about 24 h. Frozen long itud inal and tran sverse sectio ns were mounted in 50 070 aque ous glycerine and examined in a Leitz fluorescence micro scope . When stud ied in a fluore scence microscope, equipped with prop er filter combinations (8,12), EBA elicits a bright red-yellow fluorescence, while the nerve tissue elicits a green autofluorescence. Leakage of albumin through the walls of intraneural microvessels can hereb y be easily detected.
H orseradish peroxidase. Horseradish peroxidase (MW 42.000) (HRP) has been used as a tracer in num erous investigations to study the permeabilit y of intraneural microvessels and the perineurum . This substance can be detected by light microscopic, as well as ultrastructural, techn iques. In the present study HRP (400 mg/kg of body weight) was slowly injected into a tail vein or via a catheter in the femoral vein of the contralateral leg and allowed to circulate for 30 min. The vascular system of the rats was then rinsed by transcardi al perfusion with physiological saline, and fixation was performed with 3 070 cacodylate-buffered gluta raldehyde. Segments of the sciatic nerve of the vibrated leg were then removed, fixed for 24 h in 3 070 glutaraldehyde, and tran sferred to a 7.5 070 sucrose solution. Sections were treated with diaminebenzodine and hydrogen peroxidate for light microscopic demonstration of the peroxidase. HRP was easily dete cted as a brown -red substa nce, in contrast to the white color of th e nerve tissue.

Macroscopic appearance
The ne rve trunks of the vibrated leg we re surrounded by a gelatinous edema (figure 1), which appeared generally sp r ea d in the subcutaneous space. The normal st riated appearance of the superficial layer of the nerve had di sappeared (figures I and 2), the nerve exhibiting an opaque homogeneous surface indicating ep in eurial edema.

Evans blue-albumin
In the vibrated leg of five rats, fluorescen ce microscopy re vealed a diffuse red fluorescence confined to the epineurial tissue indicating protein leakage and ep ine ur ia l edema (figure 3). In contrast, the nerve fibers inside the fascicles exh ibited a normal gr een autofluorescen ce, and no red fluorescence (EBA) co uld be detected outside the vessels in the endoneurial space.

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In the contralateral limbs (controls) neither the epineurium nor the endoneurium showed any signs of leakage of the dye complex, ie, the EBA was confined to the vessel lumina in both tissue layers (figure 4).

Horseradish peroxidase
In the light microscopic analysis in the case of the five rats which received HRP, HRP could be found confined to the lumen of the endoneurial capillaries. There was, however, a leakage of the substance through the walls of the epineuriai vessels in nerves in the vibrated limb ( figure 5), where HRP could be traced diffusely in the epineurial connective tissue ( figure 6). The picture was consistent with slight epineurial edema. There were no detectable amounts of HRP in the epineurium of the sciatic nerve of the contralateral leg.

Discussion
The structure and function of the intraneural microvessels normally, and under certain pathological conditions, have been extensively outlined in numerous previous studies (8,9). There are well developed vascular plexuses in all layers of the nerve. Anastomoses between these vascular plexuses are extensive, and the nerve has a significant "buffer capacity" to compensate for the effects of local vascular trauma. The endoneurial capillaries possess a "blood-nerve barrier" corresponding to the "blood-brain barrier" of the central nervous system. It prevents macromolecules in the blood from reaching the endoneurial space. This barrier, together with the diffusion barrier in the perineurium (12,18), serves to maintain a specialized intrafascicular environment, optimizing nerve fiber function. The epineurial vessels do not possess such a diffusion barrier and react more easily to trauma by increasing their permeability. Epineurial edema is therefore seen at an early stage following ischemic, mechanical, and chemical trauma (8), while increased permeability of endoneurial vessels is seen first at a later stage in such instances.
In the present experimental model the vibration trauma induced epineurial edema. Such an edema might, in itself, interfere with nerve fiber nutrition by causing a "backward failure" in the nerve, including venular stasis and impaired oxygen supply to the nerve fibers. Hyperirritability of fibers, based upon hypoxia, results in paresthesias and numbness of the fingers. Clinically, the nocturnal, rapidly reversible paresthesias seen in carpal tunnel syndrome probably correspond to a nocturnal occurrence of such an intraneural edema, which, during the day is drained parallel to disappearance of the symptoms. If the edema becomes permanent, the symptoms also become permanent. A chronic epineurial edema may be invaded by fibroblasts and transformed into an intraneural constricting scar. In su ch in stances the symptoms are persistent and may, ultimately, be associated with nerve fiber loss and muscle wasting.
Clinical experience shows that, when symptoms simi la r to those seen in patients with carpal tunnel synd ro m e occur in male patients, the condition is often asso ciated with a long-term use o f hand-held vibrat ing tools. However , sever a l of these cases -in contrast to true carpal tunnel syndrome -do not present any nerve conduction abnormalities in the median nerve at the level of the carpal tunnel. We hypothesize that the symptoms in these instances are based upon intraneural ed ema in distal branches of th e median nerve at the level of the fin ger pulps. The edema has been in itiated by vib ratio n exposure and aggravated during the ni gh t. The specialized anatomy o f the finger pulps is characterized by numerous strong sep t um formations, constituting walls of tissue compartments. During the night the horizontal position of the body, as well as the absence of a muscle pump , contributes to an increase in tissue pressure in all ti ssue compartments of the upper extremity, including the co m p ar tments of the fing er pulps . A nocturnal inc reas e in tissue pressure in such compartments co uld be expected to affect terminal, edematous nerve branches and thereby el icit paresthesia wit hin skin a reas innervated of the median nerve. The clinical picture may imitate th e ca rp a l tunnel sy nd ro m e, but , if the m edian nerve is not involved at the more proximal carp a l tunnel level , nerve conduction is normal across th e wrist, and de compression by sp litt ing th e carpal ligament could no t be expect ed to cure the condition.