Male Sprague-Dawley rats [Hla:(SD) CVF] (Hilltop Lab Animals, Inc., Scottdale, PA) that were 6 wk of age and weighing approximately 250 g at arrival were used in both studies. Rats were maintained in a colony room with a 12:12-h light:dark cycle (lights on 0700 h) and with Teklad 2918 food and tap water available ad libitum, at the National Institute for Occupational Safety and Health (NIOSH) facility, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Rats were acclimated to the facilities for 1 wk before being used in experiments. All procedures were approved by the NIOSH Animal Care and Use Committee and were in compliance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals and the NRC Guide for the Care and Use of Laboratory Animals.

Procedures and equipment used to expose rats to vibration have been previously described (Krajnak et al., 2006; Welcome et al., 2008). Briefly, on the day of the experiment, rats were restrained in Broome style restrainers and placed in sound-attenuating chambers. Rats exposed to vibration had their tails secured to platforms attached to shakers and were exposed to vertical, sinusoidal vibration at a frequency of 125 Hz and a constant acceleration of 49 m/s² root mean square (r.m.s.) for 4 h (n = 6–8/group). Control rats also were restrained but had their tails secured to platforms mounted on isolation blocks (n = 6–8/group). Rats were exposed to vibration or control conditions and euthanized 1, 2, 7, or 9 d after exposure by exsanguination under pentobarbital anesthesia (100 mg/kg, ip). Additional groups of rats underwent the same exposure and were allowed to recover for similar amounts of time (n = 6–8/group). However, these groups of rats were exposed to another bout of vibration and euthanized 1 h after the second exposure.

After euthanasia, the C13–15 region of the tail was dissected and the ventral tail artery was immediately removed. The artery was bisected and two segments were immediately frozen in separate cryovials in liquid nitrogen. Nitrate/nitrite (NO_x) concentrations were measured in the distal segment using the NO_x colorimetric assay (Caymen Chemical Company; Ann Arbor, MI). Both assays were performed using the manufacturer's instructions. Protein concentrations were measured using the BCA protein assay (Pierce, Rockford, IL).

The remaining portion of the tail was placed in cold Dulbecco's modified medium (DMEM, Invitrogen, Carlsbad, CA) and kept at 4°C until used in microvessel studies. Ventral tail arteries from approximately the C16–20 region were dissected and the proximal section was used to measure vasoconstriction induced by the α-2C-adrenoreceptor agonist, UK14304. The distal segment was used to examine vascular re-dilation to ACh after constricting the artery with the α-1 adrenoreceptor agonist phenylephrine (PE). All vasoactive factors were purchased from Sigma Chemicals (St Louis, MO USA). Re-dilation after constriction was assessed because ventral tail arteries have little endogenous basal tone (Krajnak et al., 2006). To assess responses to vasoconstricting and dilating factors, artery segments were mounted on pipettes in a microvessel chamber (Catamount Research and Development, Living Systems, St. Albans, VT) containing HEPES buffer with glucose (10%) and sodium bicarbonate (Krajnak et al., 2006), and maintained at 37°C. Arteries were pressurized to 60 mm Hg and allowed to equilibrate for at least 1 h. The chamber buffer was then changed and vasoconstricting factors were added in half-log increments. Changes in the internal diameters of arteries were measured when arteries stabilized (approximately 5 min between applications of the agent) using an XC-ST30 video camera mounted on a Nikon T1-SM inverted microscope, a video dimension analyzer (Catamount Research and Development), and Data-Q Instruments software (Akron, OH). Re-dilation in response to ACh applied in half-log increments was measured in PE-constricted arteries and changes in the internal diameter were determined as described above. Dose-response curves to the various vasoactive factors were generated by averaging dose-dependent responses within a group and using GraphPad (Prism 5.1; San Diego, CA).

Data from rats exposed to a single bout versus two bouts of vibration were analyzed separately. Separate comparisons were also made from samples collected after varying lengths of recovery. Mean concentrations of NO_x (pm/μg protein) were calculated for each group and then analyzed using one-way analysis of variance (ANOVA). Pairwise comparisons were made using Student's t-test. The percent change in vascular diameter from baseline was calculated after the application of each dose of UK14304 or ACh. The average percent change in diameter at each dose was calculated and analyzed using two-way repeated-measures ANOVA (vibrated or control vs. dose). Significant interactions were made using oneway repeated-measures ANOVA, and Student's t-test for pairwise comparisons. All data were analyzed using JMP (version 10, SAS Institute, Inc., Cary, NC). Significant differences were those with p < .05.

The baseline diameters of arteries in rats from the four different groups were not different (mean diameter ± SEM; 1-d recovery: control 320.57 ± 20.79, vibrated 352.71 ± 19.61; 1-d recovery and reexposure: control 357.71 ± 13.59, vibrated 363.43 ± 20.22). However, after 1 d of recovery, arteries from vibrated rats displayed a significant increase in sensitivity to UK14304-mediated vasoconstriction. ACh-induced re-dilation was similar in arteries from both groups of rats (Figures 1A and 1C, respectively) Arteries from rats re-exposed to vibration after 1 d of recovery did not show a marked change in responsiveness to UK14304, but did display a reduced responsiveness to ACh-mediated re-dilation (Figures 1B and 1D, respectively). Changes in vascular responsiveness were associated with reductions in vascular NO_x concentrations (Figure 2).

After 2 d of recovery, there was no marked change in UK14304-induced vasoconstriction in arteries from rats exposed to vibration (Figure 3A). However, the reduced responsiveness to ACh-induced re-dilation was maintained in all vibration-exposed arteries (Figure 3B). NO_x concentrations also were lower in arteries collected from rats exposed to vibration than control rats (Figure 4).

Both UK14304-induced vasoconstriction and ACh-induced vasodilation were similar in control and vibrated rats after 7 d of recovery (Figures 5A and 5C, respectively). There also were no changes in vascular NO_x concentrations (Figure 6). Although re-exposure to vibration after 7 d of recovery did not markedly affect UK14304-induced vasoconstriction, there was a resultant decreased responsiveness to ACh-mediated re-dilation (Figure 5B and 5D, respectively). This change in responsiveness to ACh was associated with a reduction in vascular NO_x concentrations in revibrated arteries (Figure 6).

Arteries examined from animals following 9 d of recovery or 9 d of recovery and vibration did not display significant changes in UK14304-induced vasoconstriction (Figures 7A and 7B, respectively). Although there appeared to be an enhanced responsiveness to ACh-induced re-dilation after 9 d of recovery or recovery and vibration, the dose-dependent changes in artery diameter were not significant (Figure 7C and 7D, respectively). However, vascular NO_x concentrations were increased in arteries from both vibrated groups after 9 d of recovery (Figure 8).