Effects of low- and high-frequency local vibration on the occurrence of intimal thickening of the peripheral arteries of rats.

Effects of low- and high-frequency local vibration on the occur rence of intimal thickening of the peripheral arteries of rats. Scand J Work Environ Health 14 (1988) 312-316. Rats were exposed to local vibration in a study of the differences in the effects between low and high frequencies of vibration on the vessel wall of peripheral arteries. The vibration was delivered at frequencies of 30 and 480 Hz under a constant acceleration of 5g. The duration of the vibration ex posure was 30 d. Thechanges in the arteries werestudied pathohistologicallyand hematologically. Three of the five rats exposed to 30 Hz and three of the five rats exposed to 480 Hz showed disruption of the internal elastic lamina. The disruption was followed by focal cell proliferation with regenerative forma tion of collagen and elastic fibers. The vascular changes observed after vibration exposure could not be explained by changes in plasma lipid concentrations. These results suggest that not only low frequencies of vibration, but also high frequencies have harmful effects on the intima of small arteries.

Prolonged exposure to hand-arm vib rat ion produces Raynaud 's ph enomenon, which is o ne of th e ma in symptom s of the vibra tion syndrom e. The mechanism leading to Raynaud's phenomenon in vibration syndrome is not yet well understood. So far , conclusive evidence for this mechanism is lacking.
Some inve stigators (2,5,9) ha ve reported histologica l chan ges in the peripheral arteries o f workers who had used vibrati ng tool s and had experienced Ra ynaud 's phenomenon. It is necessary , ho wever, to ascertain exp erimentally whe ther or not th ese chan ges ar e specific to expo sure to local vibratio n. Recentl y, we (7) demonstrated experimentally the occurrence o f intimal th ickening in peripheral arteries o f rats after 90 d of exposur e to local vibration (60 H z). It is also important in the investigation of vibration hazards to determine the frequencies which may have serious effect s on the arteries. The purpose of th e pre sent study was to de term ine the differ ences in the effects of low and high frequencies of vibra tion on the occ urre nce of intimal thi ckenin g in th e peripheral arteries of rats after 30 d o f exposure to loc al vib ration.

Material and methods
Twenty-one male Wi star ra ts, initially weigh ing 230-250 g, were used in groups of five or six.
Th e animals were placed prone in individ ua l mesh cages . Th eir hin d legs were outside th e cage , and th e planta r surfaces were horizontall y fixed to th e vibrating plate by mean s of double-sided adhesive tap e so th at the vibrat ion would be transmitt ed only to the hind legs. The pa rt of th e cage co ntai ning the rest o f the animal was fixed o n a nonvibr ating plate separated fro m the shaker. Th e hind legs of the rats were exposed to vertical sinusoi dal vibrat io n with frequen cies of 30 Hz and 480 H z under a con stant acceleration o f 5g.
The duration o f exposure was 4 hid for 30 d. Th e contr ol animals for each experiment were also pla ced in the sam e po sition as th e expo sed anima ls in wire mesh cages, whic h were pla ced nea r the elect romagnet ic sha ker during the exposure exper ime nt 4 hid for 30 d so that an y no ise pro d uced wo uld be experienced but not th e vibratio n .
Before the expe riments the rats were trained for o ne week to ride o n the vibrating plat form .
Th e temperature in the lab orato ry room was kept at 18 to 22°C and a 12-h "light-dark" cycle wa s mainta ined in the animal room. Food and water were provided freel y eac h day before and after the per iod of vibra tio n.
Eightee n hou rs after th e last exposur e, blo od was co llected int o hep arin ized tubes aft er the rats were decap itated. T he blood was used for measur ing th e who le blood viscosity, hematocrit , and plasm a lipid For the microscopic examination, cross -sections of the foot, 1.5 ern per ipheral from the ankle, were obtained and were stained with hematoxylin-eosin and elastica-Van Gieson.
Whole blood visco sity was measured with a WeIIs-Brookfield cone plate microviscometer (model LVT) at 37°C at two shear rates, 115 and 230 s'.
The pla sma concentrations of to tal cholestero l and HDL cholesterol were mea sured by enz ymatic methods; the pla sma concentrations of lipoperoxide were determined with the fluorometric method developed by Yagi (11).
The statistical significance of the results was tested with Student's t-test .

Results
After 30 d of exposure, almost the same histological change s were observed in the small arteries at the exposed site in three of five rat s exposed to 30 Hz and in three of five rats exposed to 480 Hz . The changes were observed only in the intima. Namely, neither the media nor the adventitia showed any histological change. Figure I shows the histological changes in the artery of the hind leg o f a rat after exposure to 30 Hz for 30 d; disruption of internal elastic lamina and focal cell proliferation with formation of collagen and elastic fibers can be ob served . These alterations were not observed in the rats of the control groups (figure 2).
Tab le I shows the effects of local vibration exposure on the whole blood viscosity measured at shear rates of 115 and 230 S-1 an d on the hematocrit level. There were no significant differences in the levels of who le blood viscosity or hematocrit between the controls and the exposed groups at any frequency of exposure. Table 2 shows the effects of local vibration exposure on the plasma lipid concentrations. There were no significant dif ferences in the plasma concentrations of total cholesterol , HDL cholesterol or lipoperoxide between the controls and the exposed gro ups at any frequenc y of exposure .

Discussion
Take uchi & Imanishi (9) reported that intimal thickening was observed in skin biopsies of the fingers of Table 1. Whole blood Viscosity, measured at shear rates of 115 and 230 S ' l , and hematocrit levels of rats whose hind legs were exposed to vibration (30 or 480 Hz) for 4 hid for 30 d and o f their controls.

Exposure group 30 Hz
Con t rol rats Vibrat ion-exposed rat s 480 Hz Contro l rats Vibration-expose d rats only a few workers who used vibrating tools and who also experienced Raynaud's phenomenon. On the other hand, Ashe & Williams (2) reported that vibrationexposed workers with intimal thickening in skin biopsies suffered from a more severe degree of Raynaud's phenomenon than those without intimal thickening.
In the present study, we found that vibration exposure for only 30 d produced intimal thickening in the small arteries of the hind legs of rats. Therefore, the data from the present study suggest that there is some relation between intimal thickening in small arteries and attacks of Raynaud's phenomenon. In addition, our results support the theory proposed by Ashe & Williams (2) that intimal thickening is related to the severity of the attack.
In our experiment, the total cholesterol, HDL cholesterol and lipoperoxide concentrations in plasma, which are considered to be related to atherosclerosis (12), were not significantly different between the exposed and control groups at the two frequencies of vibration studied. These results suggest that there may be no relation between plasma lipid concentrations and the intimal thickening observed after vibration exposure. Therefore, we presume that the intimal thickenings observed in our experiment were very likely caused by the vibration exposure, and they have no relation to the atherosclerotic changes that are induced by the elevation of plasma lipids.
The whole blood viscosity did not change in our experiment with any frequency of vibration after 30 d of exposure. Fujinaga (3) reported that, in workers who used vibrating tools and experienced Raynaud's phenomenon, the whole blood viscosity was significantly higher than that in normal workers. We (7) previously reported that the whole blood viscosity was significantly increased, and a complete stenosis of the lumen of the small artery was observed after 90 d of exposure at 60 Hz. Therefore, there may be some relation between the increase in whole blood viscosity and the duration of vibration exposure and/or between the increase in whole blood viscosity and the severity of intimal thickening.
It is important to clarify the role of vibration frequency in the pathogenesis of vibration hazards. Earlier we (7) observed no histological change in the intima of the small arteries after 30 d of exposure at 60 Hz, 5g. However, in our present experiment, we found that intimal thickening in the small arteries of rats was induced by exposure not only to 30 Hz, which is a lower frequency than 60 Hz, but also to 480 Hz, which is a higher frequency than 60 Hz. These results suggest that considerably lower frequencies, as well as higher frequencies, of vibration can cause intimal thickening of small arteries. In addition, as has already been described, we (7) reported that intimal thickening in the small arteries was observed after 90 d of exposure at 60 Hz, 5g. Therefore, effects of exposure to 30 Hz and 480 Hz on the arterial intima were in-duced by a shorter period of exposure than those produced by 60 Hz.
Hunter et al (6) reported that Raynaud's phenomenon occurred the most frequently among workers using vibrating tools with a frequency range of 33-50 Hz. Agate & Druett (I) have suggested that the frequency range of 40-125 Hz is possibly the most hazardous in the genesis of Raynaud's phenomenon. On the other hand, Gerbis et al (4) considered the frequency range from 280 to 600 Hz as the most dangerous. Therefore, our present study and past studies (I, 4, 7) indicate that not only low frequencies, but also high frequencies of vibration can be potential etiologic factors for the occurrence of Raynaud's phenomenon.
Ross et al (8) reported that repetitive injury to the endothelium is important in the production of intimal thickening. Therefore, from our present results, one can assume that repetitive vibration exposure causes endothelial damage. Recently, Vanhoutte (10) proposed a new hypothesis stating that endothelial cells play a major role in the vasodilatation evoked by a number of neurohumoral substances, such as acethylcholine, substance P, catecholamines, etc. According to this hypothesis, the absence or dysfunction of the endothelium would favor the occurrence of abnormal vasoconstriction (vasospasm). We assume from this hypothesis that the dysfunction of vasodilation based on the endothelial damage caused by vibration exposure may one be of the etiologic factors of the occurrence of Raynaud's phenomenon in vibration syndrome.