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. Author manuscript; available in PMC: 2017 Nov 1.
Published in final edited form as: J Stroke Cerebrovasc Dis. 2015 Dec;24(12):2759–2773. doi: 10.1016/j.jstrokecerebrovasdis.2015.08.007

Cumulative Brain Injury from Motor Vehicle-Induced Whole-Body Vibration and Prevention by Human Apolipoprotein A-I Molecule Mimetic (4F) Peptide (an Apo A-I Mimetic)

Ji-Geng Yan *, Lin-ling Zhang *, Michael Agresti *, Yuhui Yan *, John LoGiudice *, James R Sanger *, Hani S Matloub *, Kirkwood A Pritchard Jr †, Safwan S Jaradeh ‡, Robert Havlik *
PMCID: PMC5664147  NIHMSID: NIHMS915429  PMID: 26433438

Abstract

Background

Insidious cumulative brain injury from motor vehicle-induced whole-body vibration (MV-WBV) has not yet been studied. The objective of the present study is to validate whether whole-body vibration for long periods causes cumulative brain injury and impairment of the cerebral function. We also explored a preventive method for MV-WBV injury.

Methods

A study simulating whole-body vibration was conducted in 72 male Sprague-Dawley rats divided into 9 groups (N = 8): (1) 2-week normal control; (2) 2-week sham control (in the tube without vibration); (3) 2-week vibration (exposed to whole-body vibration at 30 Hz and .5 G acceleration for 4 hours/day, 5 days/week for 2 weeks; vibration parameters in the present study are similar to the most common driving conditions); (4) 4-week sham control; (5) 4-week vibration; (6) 4-week vibration with human apolipoprotein A-I molecule mimetic (4F)-preconditioning; (7) 8-week sham control; (8) 8-week vibration; and (9) 8-week 4F-preconditioning group. All the rats were evaluated by behavioral, physiological, and histological studies of the brain.

Results

Brain injury from vibration is a cumulative process starting with cerebral vasoconstriction, squeezing of the endothelial cells, increased free radicals, decreased nitric oxide, insufficient blood supply to the brain, and repeated reperfusion injury to brain neurons. In the 8-week vibration group, which indicated chronic brain edema, shrunken neuron numbers increased and whole neurons atrophied, which strongly correlated with neural functional impairment. There was no prominent brain neuronal injury in the 4F groups.

Conclusions

The present study demonstrated cumulative brain injury from MV-WBV and validated the preventive effects of 4F preconditioning.

Keywords: Brain injury, neural functional impairment, whole-body vibration, neuronal atrophy

Introduction

Modern industrial and technical developments have brought many benefits and much convenience to our daily life; however, when we enjoy these benefits, we may at times overlook insidious harm. Motor vehicles (MVs), the most common form of transportation, produce whole-body vibration (WBV). Although engineers and industrial scientists have made advances to reduce WBV, their major goal is to make drivers and passengers more comfortable sitting. WBV still exists. Hand–arm vibration syndromes and disorders induced by hand–arm vibration tools have been extensively studied, but WBV injury and treatments have not yet been investigated. In 2011, 32,367 people were killed in an estimated 5,338,000 police-reported motor vehicle accidents (MVA), and 2,217,000 people were injured. A recent study by Charlie Klauer of the Virginia Tech Transportation Institute Center for Vulnerable Road User Safety states that physical fatigue is a cause of 20% of all U.S. automobile crashes. People believe tired drivers are deadly drivers. However, our recent preliminary study results1–3 on simulated motor vehicle-induced whole-body vibration (MV-WBV) challenge the traditional thinking; our studies suggest that “driver’s fatigue” is actually brain dysfunction and brain impairment resulting from MV-WBV. Here we hypothesize that prolonged MV-WBV induces brain injuries that compromise a driver’s judgment and reactive capabilities and may be one cause of MVAs. Prevention of early neural injury from MV-WBV can avert or reduce late chronic brain diseases. In our previous study on hand–arm vibration injury, human apolipoprotein A-I molecule mimetic (4F), an apo A-I mimetic, was studied, and its ability to prevent hand–arm vibration injury was validated.4 The goal of the present study was to discover the pathological process of such brain injury, to elucidate the cellular and molecular mechanism from MV-WBV injuries, and to determine whether 4F prevents WBV injury. The rat’s anatomy and physiological and biological features are similar to those of a human being.5–8 A simulated animal study is the only way to reach these objectives.

Materials and Methods

Ethics Statement

For the care and use of laboratory animals, all protocols of the present study conformed to the National Institutes of Health guidelines and received approval from the Biomedical Resource Center and the Institutional Animal Care and Use Committee at our institution (AUA-2363). After the animals arrived, they were allowed to acclimate for 7 days before exposure. The animals were housed in a central animal care facility with 12-hour light cycles and were given food and water ad libitum.

Animal Study Model and Vibration Setup

Animal Groups

Seventy-two Sprague-Dawley male rats (weight 250–300 g) were divided into 9 groups (N = 8)1: the 2-week normal control group had no treatment2; the 2-week sham control group was restrained in the tube without vibration3; the 2-week vibration group was exposed to WBV at 30 Hz and .5 g acceleration for 4 hours/day, 5 days/week for 2 weeks4; the 4-week sham control group was restrained in the tube without vibration5; the 4-week vibration group was vibrated for 4 weeks6; the 4-week vibration group was treated with 4F-peptide (Ac-DWFKAFYDKVAEKFKEAF-NH2) preconditioning7; the 8-week sham control group was restrained in the tube without vibration8; the 8-week vibration group was vibrated for 8 weeks9; and the 8-week vibration group was treated with 4F-peptide preconditioning. At the end point, all the rats were evaluated by behavioral, physiological, histopathological, and molecular studies of the brain.

Vibration Setup

The rats in all the groups were placed individually in polyvinyl chloride tubes. Rats were given 1 day training to adapt to this small tube space, after which all the animals voluntarily entered the tubes throughout the experiment.

The rat exhibited no stress at all as seen in our previous rat experiments.8 Actually, in all groups the rats were very still and calm for most of the time while in the tubes. The tubes were taped to a vibrating platform and the tails were taped alongside the tube on the platform (Fig 1, A). Vibration was performed without any sedative or anesthesia. The electromagnetic vibration motor (type 4809; Brüel & Kjær [B&K], Skodsborg, Denmark) was driven by a sine wave signal from a function generator (Simpson 420; Simpson Electric Co., Elgin, Illinois). The acceleration was set with a power amplifier (B&K type 2706). Frequency and acceleration was calibrated before beginning the study using an HP 1201 B oscilloscope and a B&K 4384 accelerometer connected to a B&K Integrating Vibration Meter, type 2513, linear vertical oscillations with linear vertical oscillations of 30 Hz and .5 g acceleration (4.9 m/second2 root mean square acceleration). These vibration parameters were selected to simulate most common MV driving.10–18 The most common frequency range of WBV from MVD that directly affects the human head is from 20 to 32 Hz.19,20 The reliability of this animal model for vibration studies has been identified and compared to other vibration animal models, demonstrating by systematic and continued studies that this model is psychologically stress-free.8

Figure 1.

Figure 1

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(A) Whole-body vibrating setup. Rat voluntarily enters a PVC tube taped to a vibrating platform. (B) Maze test. Square size is 23 × 23 inches, the channels are 4 inches wide. A rat enters in the lower left corner; the exit is in the upper right corner with food. Abbreviation: PVC, polyvinyl chloride.

Conditioning Rats with 4F

The 4F (amino acid sequence: Ac-DWFKAFYDKVAEKFKEAF-NH2) was synthesized by the Blood Center of Wisconsin. The peptide was reconstituted in normal sterile saline. The preconditioning group received subcutaneous injections of 4F (3 mg/kg) every day approximately 30 minutes before the daily exposure. Rats were weighed daily to adjust the dosage of 4F as required. The rationale for the administering paradigm of 4F to the rats was as follows: Oral dosing in the food or water would not provide enough accuracy; therefore, we administered 4F as a subcutaneous injection to verify the exact amount given to each rat every time. The dosing concentration of 3 mg/kg has been shown to be quite effective in reducing the effects of vibration injury as found by our previous study.4 It is similar to the concentration used in human studies and is at the lower end of other published in vivo studies using 4F.4,21–25 The published 4F half-life times vary from 6 to 12 hours. Our hypothesis for the timing of injections is that preconditioning might be most important because the initial WBV effect is vasospasm and 4F is a strong vasodilator and neural protector, as shown in our previous published work of vibration study. Having a high circulating concentration of 4F at the onset of vibration might prove to be the best prevention approach. In addition, a recently completed clinical trial titled: “4-EVER: a Trial Investigating the Safety of 4F Endovascular Treatment of Infra-Inguinal Arterial Stenotic Disease” (ClinicalTrials.gov Identifier: NCT01413139) has shown positive results.26

Evaluation Methods

General Observation and Behavior Observation Using a Maze Test

Vital signs, including heart rate, respiratory rate, blood oxygen saturation, food intake, weight gain, hair appearance, and so on, were observed daily. A maze test was used to evaluate the rat’s memory and judgment capability. We constructed a classic maze used in our previous study on WBV.1,2 The maze is constructed from standard ½-inch particle board and consists of a large 23 × 23-inch platform with a series of 6-inch high vertical walls and a transparent ceiling. The channels are 4 inches wide, allowing the rats easy access through the maze. We made a modification to the lower right corner to make it slightly more difficult. The rat started at the left lower corner entrance, ran through the maze, and finished at the right upper corner exit, with a food reward (“Fruit Loops”). Each rat was run through the maze twice each day following the vibration or other treatment. These runs started during the final week of vibration, so 2 tests/day × 5 days = 10 time tests were done for each rat. We recorded the times and number of errors. The times and number of mistakes were video-recorded to determine if there had been changes in brain function. Prolonged time and increased error times indicated brain dysfunction or impairment.27,28

Tests for Sensory and Motor Functional Impairment from MV-WBV

Tail-Flick Test

Thermometric Sensory Test

After WBV, while the rats were still in the tubes, we measured the time it took the tail to withdraw from a heat stimulus. The rats were placed near the tail-flick apparatus (Model TF-6, Emdie Instruments Co, Maidens, VA) and the tail was placed into the groove of this apparatus. The time it took the rat to withdraw the tail was read and recorded. Delayed time indicated injury to the tail nerve from WBV.29

Von Frey Filament Test

We used a dynamic plantar aesthesiometer (Ugo Basile, Comerio, Italy) to perform a touch sensory test. Rats exhibit a hind paw withdrawal reflex whenever they perceive that their paw has been touched. This device uses a filament to measure the upward force and time required for the rat to withdraw its paw after it is touched. When the aesthesiometer is engaged, the tip of the filament rises and presses against the skin of the rear paw at right angles. Force of application is increased over time. The researcher records the time and force required for a paw withdrawal.30,31 Increased response time or force indicated injury of the sciatic nerve from WBV.

Grip-Strength Test

We assessed motor function with a rat grip-strength meter (Chatillon Model 10 lbf; AMETEK, Inc., Largo, FL). This technique uses a bar attached to a force meter placed near the front limb of the subject rat. The rats were grasped by the tail and nape and placed close to the T-bar. They were coaxed to grasp the bar with both paws. The rats were drawn backward by the tail until they released the T-bar. Immediately after release, grip forces were recorded. Reduced strength in the front paw is an indication of injury to the motor fibers of median and ulnar nerves and loss of muscle tone.30,32

Blood Flow Volume Measurement by Laser Doppler

The rat’s carotid and temporal artery blood flow was measured as an index of blood supply to the brain using a GE Vivid 7 Ultrasound System and GE Echo software (GE Healthcare, Wauwatosa, WI). Rats were anesthetized using isoflurane gas (.5%–1.0% with O2 at 1 L/minute), which was kept to a minimal dose to reduce any vasodilation caused by deeper anesthesia. The same gas dose/minute was inhaled for each rat to avoid anesthetic effect on blood flow during the test. After shaving the neck and face, we gently positioned the probe on the carotid or temporal arteries to visualize the vessel lumen on the monitor screen. Three images of each artery were taken for analysis. A greater reduction means greater vasoconstriction or chronic vascular arteriosclerosis. The software automatically calculated the flow volume using the standard formula. The instantaneous flow rate can be calculated as cross-sectional area (in cm2) × flow velocity (in cm/second).33 The Doppler-computerized program automatically calculated the blood flow volume in cubic centimeters per minute.

Tissue Processing and Histopathological Studies

Tissue Harvesting and Processing

Light microscopic (LM) and transmission electron microscopic (TEM) studies were carried out on the middle cerebral artery (MCA) and its opposite brain cortex. At the end time point, the rats were anesthetized using isoflurane gas inhalation. Rat skull was carefully opened using a microsurgical saw under a surgical microscope. On the right side, the right MCA and its opposite brain cortex (1-mm thickness) were carefully harvested using a #11 blade without any traumatic intervention. Specimens were immediately immersed into a 2.5% glutaraldehyde solution in phosphate-buffered saline. They were then processed routinely and embedded in a mixture consisting of 76.0 g Medcast (Ted Pella Inc., Redding, CA), 18.0 g Araldite 502 (Ted Pella Inc), 39.0 g dodecenyl succinic anhydride (DDSA), 61.0 g nadic methyl anhydride, and 2.0%–3.5% 2,4,6-Tris(dimethylaminomethyl)phenol-30 by volume. Semithin transverse sections (.5 μm) were cut and stained with toluidine blue for the LM study. Ultrathin sections (50–70 nm) of the arteries and the brain cortex stained with salts of uranyl acetate and lead citrate were used for the TEM study. In the semithin sections, the vasodilation degree (EC/EM ratio) was calculated by measuring the endothelial circumference (EC) and the internal elastic membrane (EM) length and dividing them (EC/EM). The squeezing of the endothelium and a curved and unchanged EM length indicated a lumen size change. This is a proven accurate measurement.34,35 A lower mean EC/EM ratio means a smaller lumen, and the vasoconstriction in the acute WBV stage may become arteriosclerosis in the chronic WBV stage. Artery samples for measurement of vascular wall thickness and fibrosis observation were also obtained. On the left side, the left MCA and its opposite cortex tissue were harvested in the same way. The brain membrane was kept intact to avoid artifact neuronal injury and was immediately immersed in the 10% formalin in .1 M phosphate-buffered saline at pH 7.4. The tissues were then processed for routine paraffin embedding. Blocks were stored at 4°C until they were sectioned for hematoxylin and eosin (H&E) stain and for measurement of superoxide anions (O2−) in the brain tissue.

Neuronal Pathological Analysis

General Observation of the Sections of Cortex

Four kinds of histopathological sections were prepared for the present study: (1) Semithin sections were observed for cerebral vascular changes; (2) the H&E stained sections were observed and analyzed for neuron and glial cell changes; (3) the nitrated tyrosine (NT) sections of the brain artery and cortex were used for measurement of superoxide anions (O2−); and (4) the TEM section was observed for more detailed cellular changes.

Necrotic Dark Shrunken Neuron (DSN) Counting and Analysis

First, in each H&E section under 100× magnification, we found the MCA, moved its opposite cortex center to the central field, and then increased the magnification to 400×. Finally, the dark neurons were counted in this whole microscopic field. The commonly recognized criteria for necrotic neurons were used: pyramid shape with a visible axonal tail, dark and intense purple stain with an obscure nucleus or without a visible nucleus. Although the rats were still alive during cortex tissue harvesting, we used nontraumatic microsurgical techniques, and harvested and immersed this living tissue into the fixing solution in seconds. Artifacts may make DSNs; however, to avoid this artifact bias, the numbers of DSNs in sham control group were also counted, and comparison between the vibration group and sham control or 4F-preconditioning group was performed.

Measurement of Neuronal Nuclei Area

The neuron size became smaller after 4-week vibration; after 8-week vibration this feature became prominent in the H&E stained sections. For accurate neuron size, the nucleus area was measured, as its contour was sharply clear with deep purple-blue stain, while the neuronal edge of the plasma membrane of some neurons was not so clear because of a lighter stain. The nucleolus area of every neuron at the MCA opposite cortex area was measured using our standard procedure and MetaVue Software (Molecular Devices, Sunnyvale, CA). Each neuron in the visual field was outlined and the software automatically calculated the area of each neuron. Standard statistical analysis followed.

Evaluation for Molecular Changes after WBV

Measurement of Superoxide Anions (O2−)

The NT in the brain artery and cortex was measured. As described in our previous vibration study on a rat tail model,4 WBV injury produces superoxide anions. These anions combine with nitric oxide (NO) to form peroxynitrite, which nitrates tyrosine residues in the proteins of the smooth muscles. We tested for these nitrotyrosine residues. On the left side, MCA and the opposite cortex tissue were harvested and processed, and paraffin was embedded (see the above section). The sections were cut at 6 μm for immunohistochemistry. The sections then were deparaffinized with 2 100% xylene rinses (5 minutes for each rinse) followed by progressive rehydration with phosphate-buffered saline. Sections were blocked (to remove nonspecific binding) with 2% normal goat serum, 20% avidin in phosphate-buffered saline for 60 minutes before indirect immunoperoxidase staining for NTs by incubating with rabbit antinitrotyrosine and 20% biotin overnight at 4°C (1:250; Upstate Biotechnology, Lake Placid, NY) followed by goat antirabbit IgG for 60 minutes room temperature (RT) (1:200; Vector Laboratories, Burlingame, CA). Superoxide combines with NO to generate peroxynitrite, which nitrates tyrosine residues of proteins to form nitrotyrosine. Peroxidase staining was developed with a Vector kit (Vector Laboratories). Images of the immunostained cross sections of whole arteries/brain tissues were captured digitally with a Zeiss AxioVision light microscope (Zeiss, Oberkochen-Königsbronn, Germany). To quantify immunoperoxidase staining, sections from all group arteries/brain tissues were incubated together and photographed digitally at the same exposure and light intensity setting and ×200 magnification. The optical density of staining was analyzed using MetaVue 5.0r7 software (Universal Imaging Corporation, Downingtown, PA). The mean optical densities of the arteries/brain tissues using relative intensity units (RIUs) in each group were compared and statistically analyzed. The RIUs are internally set by the program. The presence of more nitrotyrosine means a darker staining and a lower RIU value.

NO Measurement

During tissue harvest, blood was withdrawn from the common carotid artery, allowed to clot, and the serum frozen at −80°C. Several concentrations of NaNO3 solutions (2.5, 5.0, 10.0, and 25.0 μM) were used for the creation of a standard curve. The standard solutions were injected into a vanadium (III) chloride-based Sievers 280i Nitric Oxide Analyzer (IBG, Boulder, CO). The resulting numbers were used to create the standard curve to calculate the concentrations of NO3 in the plasma. Plasma samples were then injected into the analyzer and the readings recorded. Briefly, the peaks were identified and the internal software automatically calculated the area size of the peaks. Each rat plasma sample was injected several times, and then the peak area sizes were averaged. The peak area sizes are directly proportional to the amount of NO3 found in the samples. Using the NaNO3 standard curve, the average peak area sizes were converted to NO3 concentrations. Standard statistical analysis of these final NO3 μM concentrations of the plasmas followed. The concentration of NO3 is directly proportional to the amount of NO in the plasma. Therefore, the NO3 concentration directly reflects the amount of NO in the blood.36

Statistical Analysis Methods

A 2-way analysis of variance (ANOVA) test was used to compare outcomes between the following groups: sham control, vibration, and 4F-preconditioning (Tables 1, 2) at 2, 4, and 8 weeks. An interaction between group effect (sham control vs. vibration vs. 4F-preconditioning groups) and time effect (weeks) was tested. The normality of residuals was examined and all ANOVA models satisfied this assumption. Pearson correlation coefficient (r) analysis was used to measure the strength of correlation between the results of the brain functional test (maze test)/motor/sensory function with physiological and histological results (blood flow of the carotid artery/EC/EM ratio/endothelial nitric oxide synthase [eNOS]/NO), and so on. All analyses were performed in SAS 9.3 (SAS Institute Inc., Cary, NC).

Table 1.

Neural physiological impairments from WBV (n = 8)

Term Group Maze test
Von Frey Filament Test
Tail flick (s) Grip (g)
Time (s) Error (numbers) NCV (m/ms) Time (s)
2-w Normal   83 ± 21 2.1 ± 1.0   42 ± 7   20 ± 1 3.6 ± .2 1502 ± 103
Sham   88 ± 38 2.3 ± 1.0   41 ± 8   20 ± 1 3.7 ± .6 1468 ± 109
Vibration 135 ± 32 4.0 ± 2.0   30 ± 8   26 ± 4 4.8 ± .5 1042 ± 171
4-w Sham   91 ± 25 1.7 ± .7   58 ± 7   15 ± 2 3.6 ± .2 1859 ± 84
Vibration 132 ± 10 4.2 ± .5   36 ± 4   20 ± 2 5.0 ± .4 1611 ± 186
4F-Prc 109 ± 15 2.0 ± .5 52.8 ± 5    14.7 ± 2.6 4.0 ± .3 1817 ± 99
8-w Sham 122 ± 22 1.2 ± .4   59 ± 5   15 ± 2 3.7 ± .1 2108 ± 104
Vibration 178 ± 5   4.6 ± .4   33 ± 4   22 ± 2 5.3 ± .5 1720 ± 93
4F-Prc 131 ± 31 1.5 ± .6   54.5 ± 5.5   13.7 ± 2.5 4.3 ± .3 2066 ± 33
P values
2-w Normal vs. sham .7494 .6952 .7941 1.0       .6616 .5317
2-w* Vibration vs. sham .0181 .0495 .0159 .0013 .0014 .0001
4-w** .0007 .0023 <.0001 <.0001 <.0001 .0011
8-w** .0001 <.0001 <.0001 <.0001 .0001 <.0001
4-w vs. 8-w** Vibration <.0008 <.0001 <.0001 <.0001 .0003 <.0001
4-w** Vibration vs. 4F-Prc .0028 <.0001 <.0001 .0001 <.0001 .0097
8-w** .0008 <.0001 <.0001 <.0001 .0003 <.0001
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Abbreviations: 2-w, 2-week group; 4-w, 4-week group; 4F-Prc, 4F-preconditioning; 8-w, 8-week group; NCV, nerve conduction velocity; WBV, whole-body vibration.

*

Differences were statistically significant (all P < .05).

**

Differences were very statistically significant (all P < .01).

Table 2.

Cerebral blood flow and cellular and molecular changes after vibration

Term Group Blood flow (mL/min)
Vasoconstriction ratio (EC/EM) Nitric oxide (μM) Nitrated tyrosine (RIU)
Carotid artery Temporal artery
2-w Normal 8.2 ± .6 3.9 ± .5 .8 ± .1 n/a n/a
Sham   8.0 ± 1.1 3.4 ± .6 .8 ± .04 n/a n/a
Vibration   5.3 ± 1.1 2.3 ± .3 .7 ± .1 n/a n/a
4-w Sham 15.9 ± 1.3 6.7 ± .5 .8 ± .03 14.9 ± 2.1 101 ± 20
Vibration 13.3 ± 1.3 4.9 ± .9 .6 ± .04 8.5 ± .7   71 ± 15
4F-Prc 15.5 ± .5   6.6 ± .4 .7 ± .03 13.4 ± 2.8      99 ± 13.2
8-w Sham 19.1 ± 1    8.8 ± .8 .8 ± .05 11.6 ± 2.7 107 ± 13
Vibration    15 ± 2.7 6.1 ± .4 .6 ± .07 6.1 ± 1  55 ± 6
4F-Prc 20.5 ± 1.8 9.3 ± 1  .8 ± .03 15.4 ± 3       96 ± 4.5
P values
2-w Normal vs. sham .6586 .0917 .9797 n/a    n/a   
2-w* Vibration vs. sham .0002 .0004 .0199 n/a    n/a   
4-w** .0093 .0013 .0001 <.0001 .0044
8-w** .0003 <.0001 .0003 <.0001 <.0001
4-w vs. 8-w** Vibration .0003 <.0001 <.0001 <.0001 <.0001
4-w Vibration vs. 4F-Prc .0005 .0002 <.0001 .0003 .0014
8-w** .0003 <.0001 .0003 <.0001 <.0001
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Abbreviations: 2-w, 2-week group; 4F-Prc, 4F-preconditioning; 4-w, 4-week group; 8-w, 8-week group; EC, endothelial circumference; EC/EM, endothelial circumference/internal elastic membrane ratio; EM, elastic membrane; n/a, not applicable; RIU, relative intensity unit.

*

Differences were statistically significant (all P < .05).

**

Differences were very statistically significant (all P < .01).

Results

General Observation

The general behavior and welfare of all animals were monitored daily. No animal showed abnormal behaviors of stress such as shaking, clenching, biting, postural arching, or clawing. Health inspections included checking for secretions of eyes/nose, porphyrin discharge (chromodacryorrhea), or hematuria. No animals showed any of these ill appearances nor persistent diarrhea, weight loss, light avoidance, lethargy, head waving (inner ear infection), or congested breathing. The vital signs of all the rats, such as heart rate, respiratory rate, and oxygen saturation, were stable. The long-term (8-week) results of these measurements and comparisons were as follows: mean heart rate: sham control 330 ± 8.3, vibration 331 ± 7.2 (P = .80)/minute; mean oxygen saturation: sham control 90.1 ± 1.5, vibration 89.5 ± 3.1% (P = .63); and mean respiration rate: sham control 97 ± 5.1, vibration 99 ± 6.5 (P = .51)/minute. Eating and drinking showed no changes, and the growth rate of all rats was in the normal scope. The average 8-week weight gains were as follows: sham control 79.6 ± 14.8 and vibration 83.4 ± 16.9 (P = .64). As seen, the above comparison of the sham control with the vibration animals has no statistical significance. Also, the differences of all compared mean values between normal control groups and sham control groups were not statistically significant (all P > .5; Table 1). This intense observation has shown that this animal model is stress free, further confirming our previously published work.4,8,35 In our later studies, only the WBV factor was tested.

Results of Behavioral and Physiological Studies

Central and Peripheral Neural Impairment

For the maze test, the vibrated rats were slow moving and showed hesitation, entering the wrong way and turning around (counted as errors). Every comparison of results between the vibrated group and the sham control group, either in the 2-, 4-, or 8-week treatment groups after vibration showed the completed maze time of the vibrated rats was delayed (Fig 2, A); error numbers increased (Fig 2, B); nerve conduction velocity (NCV) was reduced (Fig 2, C); Von Frey sensory time was delayed (Fig 2, D); tail withdrawal time (flick) was extended (Fig 2, E); and grip force was reduced (Fig 2, F). The mean NCV value in the 4- or 8-week sham control group was greater than the 2-week sham control group, and the difference was statistically significant (P < .0001), which indicated that following rat growth, the NCV increased; but no statistical significance was found between the vibration groups (2 weeks vs. 4 weeks, P = .08; 4 weeks vs. 8 weeks, P = .16; 2 weeks vs. 8 weeks, P = .36), which indicated vibration impaired the NCV increase. At the same age, NCV figures in the vibration groups were obviously less than those in the sham control groups (P < .02 and P < .0001; Table 1). The injuries to peripheral nervous functional effects were prominent in the vibrated groups: 8-week tail-flick (sensory) time or VF (sensory) withdrawal time was greater than 4-week; 4-week was greater than 2-week; 8-week grip (motor) force was less than 4-week; 4-week was less than 2-week (Table 1). All the differences were statistically significant, which further indicated that the longer the vibration term, the more severe the injury.

Figure 2.

Figure 2

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All the tested results in the 8-week groups: SC, VB, and PRC. Each mean value in the VIB group was very different from the SC or PRC group for every test. All differences were statistically significant (all P < .01). (A) Average times (in seconds) for rats in each group to complete maze. (B) Average numbers of maze-running errors made by rats in each group. (C) Mean nerve conduction velocity for each group. Reduction in velocity indicates impairment. (D) Average time from touch stimulus to paw withdrawal during Von Frey filament test in each group. Increased response time or force indicates injury of the sciatic nerve from WBV. (E) Average time from heat stimulus to tail flick in each group. Delayed time indicates injury to the tail nerve from WBV. (F) Grip strength. Average force exerted by rats in each group before releasing bar. Reduced strength indicates injury to motor fibers of median and ulnar nerves. (G) Carotid blood flow for each group, expressed in cubic centimeters per minute. (Computer-calculated as cross-sectional area based on three laser Doppler images of each artery.) (H) Temporal-artery blood flow for each group, expressed in cubic centimeters per minute. (Computer-calculated from cross-sectional area based on three laser Doppler images of each artery.) This artery blood flow directly affects the optic nerve. (I) Average vasoconstriction ratio (endothelial circumference divided by elastic membrane circumference) for each group. A lower mean EC/EM ratio reveals a smaller lumen, or constriction of the vessels. (J) Relative presence of superoxide anions in each group as measured in nitrated tyrosine (NT) sections of the brain artery and cortex. These free radicals are directly harmful to neural cells. (K) Relative presence of nitric oxide in each group. Decreased nitric oxide will reduce vasoconstriction ratio. Abbreviations: PRC, preconditioning; SC, sham control; VIB, vibration.

Blood Supply to the Brain Was Reduced from WBV

The flow of common carotid and temporal arteries was decreased (Figs 2, G,H, 3). All differences between the vibration groups and the sham control were very statistically significant (all P < .0001 or P < .005; Table 2). In the present study, rats began vibration exposure at a young age (3 months old), weight at 250–300 g. As the treatment term was lengthened and the rats grew, some normal physiological values changed. These included an increase in body weight/size, brain volume, vessel size, muscle mass, and so on. We used the 2-way ANOVA test to compare measurements at 2, 4, and 8 weeks between the sham control groups and vibration groups. The statistical analysis results between 4- and 8-week vibration groups are described in detail in Tables 1 and 2. In all the tests marked with 2 asterisks (**), the differences were very statistically significant (P < .001 or less). These results showed that as the vibration term lengthened, the damage became more severe, rat growth did not compensate for the vibration injuries, and vibration damage was accumulated. Comparison between the 4-week vibration group and the 4-week 4F-preconditioning vibration group, and that between the 8-week vibration group and the 8-week 4F-preconditioning vibration group showed that all the differences of the tested results were statistically significant (all P < .0005; Table 2). The comparison between the 8-week sham control group and the 8-week 4F-preconditioning group showed that none of the differences were statistically significant, which confirmed 4F’s preventive effects.

Figure 3.

Figure 3

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Blood flow volume (mL/minute) of the common carotid artery. The top bar indicates the different study terms. Volumes are given for the following groups: SC, VB, and PRC. The mean volumes in the WBV groups were much less than those in the SC or PRC groups for each vibrated term. The differences were statistically significant (all P < .01). Abbreviations: PRC, preconditioning; SC, sham control; VIB, vibration; WBV, whole-body vibration.

Cerebral–Vascular Pathological Features in Different Vibration Terms

Vascular Pathological Changes

MCA Pathological Changes

In the 2-week vibration group, the vasoconstriction degree—EC/EM ratio—in the vibration group was less than that in the sham control or normal control (all P < .0001). The cerebral vessels started with spasm, then structural constriction; the squeezing of the endothelium and a curved and unchanged EM length indicated a reduction in the lumen size and a mild endothelial cell separation from the internal EM. The corresponding cerebral blood flow and neural functions were also compromised (Table 1).

In the 4-week group, the rat’s MCA in the sham control showed intact middle-artery structure; its endothelium, internal EM, and muscular layer were very clear (Fig 4, A). However, substantial cerebral tissue injuries had developed in the 4-week vibration group. The cerebral arteries were more constricted than in the 2-week vibrated rats; the lumen was apparently reduced; and partial endothelial cells were squeezed and peeled off. This occurred in all 8 rats. The artery wall was apparently thickened; there were many fibroblasts surrounding these arteries; and vacuoles appeared in the smooth muscle layers in the LM study (Fig 4, B). In the 8-week vibration group, the whole wall of the MCA was of uneven thickness; its cell arrangement was irregular; both the endothelium and adventitia were coarse and partially broken (Fig 4, C).

Figure 4.

Figure 4

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Cross section of the rat’s middle cerebral artery. (A) Sham control section of the 4-week group: a well-opened lumen with intact endothelium, internal elastic membrane, and smooth muscle layer. (B) A section of the 4-week vibration group: constricted lumen with a thickened wall. The white arrows indicate squeezed-out endothelial cells; the white arrowheads indicate vacuoles in the smooth muscle layer. (C) A section of the 8-week vibration group: The whole wall of the artery was of an uneven thickness; its cell arrangement was irregular; the endothelium was coarse and not intact; the black arrows indicate the loss of endothelial cells and internal elastic membrane; and the white arrowheads indicate loss of the adventitia and smooth muscle. Semithin epoxy section (.5 μm) with toluidine blue stain. Bar = 100 μm.

Brain Cortex Capillary Pathological Changes

In the 4-week sham control group, both the basement membrane of the endothelial cell and the cell membrane of the pericyte were clear and intact, and the lumen was open (Fig 5, A), while in a portion of the rats in the 4-week vibration group and in that of all the rats in the 8-week group, the brain capillaries had significant characteristic changes compared to a normal capillary or sham control: the basement membrane disappeared, and the wall lost normal tension and collapsed. Its wall also became thicker (Fig 5, B). Some surrounding tissues of the cerebral arteries and capillaries disappeared, shown as a surrounding empty area, which indicated chronic edema. All these features indicate that capillary scleroses have occurred; the lumen became reduced; and the blood perfusion of the whole brain was dramatically compromised.

Figure 5.

Figure 5

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TEM section of the cerebral capillary. (A) The 4-week sham control group: Arrows indicate the BM, which is a clear intense black line. (B) The 4-week vibration group: Its BM was invisible or disappeared. The arrowheads indicate the cell membrane of the pericyte cells, which was a clear and intense black structure in A, but became a coarse, thicker gray structure in B. The whole capillary wall is seen as thicker and more fibrotic in B than in A. Bar = 1 μm. Abbreviations: BM, basement membrane; E, endothelial cell; L, lumen; P, pericyte cell; R, red blood cell; TEM, transmission electron microscopic.

The results of the superoxide anion (O2−) measurements by NT in the brain artery and cortex showed that the mean NT values in the 4- and 8-week vibration groups were greater than those in the sham control groups (P < .005), which indicated more free radicals in the arteries of vibrated rats than those in the sham control group (Table 2, Fig 2). The comparison between the 4- or 8-week vibration group and the 4- or 8-week 4F-preconditioning group showed that all the differences were statistically significant (Fig 2, Table 2).

Results of the Neuronal Pathological Analysis

General Observation in H&E Sections of the Cortex

The neurons in the 4- and 8-week vibration groups looked smaller than those in either the sham control group or the 4F-peptide preconditioned vibration group. More dark shrunken or necrotic neurons were found in the above vibration groups than in either the sham control or the 4F-preconditioning group. Other observations were that glial cells proliferated in the 8-week vibration groups and neutrophil vacuoles started to form in the 4-week vibration group, and were more prominent in the 8-week vibration group (Fig 6, A).

Figure 6.

Figure 6

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Neuronal pathological changes of the cerebral cortex. (A) Eight-week sham control group: Black arrows indicate normal healthy neurons with clear nuclei and nucleoli; white arrowheads indicate glial cells; and black arrowheads indicate only 2 dark DSNs in this section. The star marker indicates normal capillary. (B) Eight-week vibration group: Black arrowheads indicate many typical DSNs with surrounding wide white space (edema); several DSNs are seen in this section. Black arrows indicate normal neurons; white arrowheads indicate glial cells with surrounding white spaces (edema); dotted arrows point to many white clefts in the background, which indicate wide edema existing in the neurophils; and a star marker indicates a capillary with a thickened wall and narrow lumen. (C) Eight-week 4F-preconditioning group: Black arrows indicate healthy neurons with only one DSN (shown in this section by a black arrowhead); white arrows indicate healthy glial cells without any surrounding white spaces (edema); and dotted arrows indicate healthy neurophils, shown as a solid purple background without any clefts (edema). H&E stain. Bar = 25 μm. Abbreviations: 4F, human apo-lipoprotein A-I molecule mimetic; DSN, dark and shrunken neuron; H&E, hematoxylin and eosin. (Color version of this figure is available online.)

Results of Necrotic DSN Counting and Analysis

Artifacts may produce DSNs; to avoid this artifact bias, the numbers of DSN in the sham control group were also counted (Fig 6, A), and comparison between the vibration group and the sham control or 4F-preconditioning group was performed. The mean DSN count was 5 ± 2 in the sham control group, while that in the vibration group was 27 ± 11 (Fig 6, B), which was 5.36 times higher than that in the sham control group, and the difference was statistically significant (P < .0001). The mean DSN count in the 4F-preconditioning vibration group was 5 ± 2 (Fig 6, C), which was similar to that in the sham control group, and the difference was not statistically significant (P > .5). Thus, the 4F prevention effect was confirmed (Fig 7).

Figure 7.

Figure 7

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Statistical analysis of neuronal pathological changes in 8-week study groups. (A) Comparison of the mean neuron areas. The mean nucleus area of the vibration group area was less than that of the sham control or the 4F-preconditioning group. The differences were statistically significant (all P < .0001). (B) Comparison of numbers of DSN neurons. The number in the vibration group was greater than that in the sham control group or the 4F-preconditioning group. The differences were statistically significant (all P < .0001). Abbreviations: 4F, human apo-lipoprotein A-I molecule mimetic; DSN, dark and shrunken neuron; PRC, 4F-preconditioning group; SC, sham control group; VIB, vibration group.

Results of the Measurement of the Neuronal Nucleus Area

In the 8-week groups, the mean nucleus areas of the groups were as follows: sham control, 139 ± 8 μm2 (Fig 6, A); vibration group, 114 ± 8 μm2 (Fig 6, B); and 4F-conditioning vibration, 142 ± 8 μm2 (Fig 6, C). The mean nucleus area of the vibration group was obviously smaller than that of the sham control group or the 4F-conditioning vibration group (Fig 7). All the differences were statistically significant (both P < .0001). The mean nucleus area of the 4F-conditioning vibration group was similar to that of the sham control group, and the difference was not statistically significant (P = .45).

Results of Evaluation for Molecular Changes after WBV

Measurement of Superoxide Anions (O2−)

The NT stain of the whole brain tissue in the 4-week vibration group became darker than that in the 4-week sham control group, which indicated that the free radicals of the 4-week vibration group had increased. The NT stain in the 8-week vibration group was darker than that in the 4-week vibration group (P < .05, Table 2); however, the free radicals did not increase in either the 4- or 8-week 4F-preconditioning vibration group (Figs 8, A–C, 2, J).

Figure 8.

Figure 8

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Cross section of the rat’s middle cerebral artery with NT immunochemical stain. These sections were from the 4-week study groups. A lighter staining indicated less NT and fewer free radicals. A darker staining indicated more free radicals. (A) Sham control shows a well-opened lumen with brighter NT staining indicating fewer free radicals. (B) Vibration group shows a constricted lumen with the darkest NT staining, indicating more free radicals. (C) 4F-preconditioning group is similar to the sham control group, with a lighter stain, indicating fewer free radicals compared with the vibration group. Bar = 100 μm. Abbreviations: 4F, human apo-lipoprotein A-I molecule mimetic; NT, nitrated tyrosine.

Results of NO Blood Measurement

In the 4-week studies, the mean NO concentration in the sham control group was much greater than that in the vibration group, and the difference was statistically very significant (P < .0001). Similarly, in the 8-week studies, the mean NO concentration in the sham control group was greater than that in the vibration group, and the difference was also statistically very significant (P < .0001). The mean NO concentration in the 8-week 4F-preconditioned vibration group was much more than that in the 8-week vibration control or sham control group, and both differences were statistically significant (sham control group vs. vibration group, and vibration group vs. preconditioning group, P < .0001; sham control group vs. preconditioning group, P < .05; Fig 2, K), which indicated that 4F peptide increased the blood NO concentration in the preconditioned rats. The 4F preventive effect was shown to be reliable.

Discussion

Comparison of every tested result (mean value) in the normal control group with that of the sham control group was not statistically significant (all P > .5; Tables 1, 2). These results further demonstrated this animal model to be completely stress-free. We allowed the rats to acclimate for 1 week after arrival. During this time, we placed polyvinyl chloride tubes in their cages so that the rats became used to them and marked them with their scent, which would eliminate unfamiliarity later when they were introduced to the testing apparatus. This simulates a rat housing tube. Rats like this hidden space and automatically enter it without the need for any manual help or sedative or anesthesia because it is consistent with a rat’s life habit from its evolutionary history of burrowing underground for food or safe shelters. The rat neural system is similar to the human one. Thus, the present study is a close simulation of a real human MV-WBV situation and eliminates any stress or drug side effects.

The reason we chose to study the subjects at different time points, at 2 weeks (acute), 4 weeks (short term), and 8 weeks (medium to long term), and to administer different tests was that, compared to the average rat life span of 3 years, the relative average human life span in the United States is 78 years, about 26 times that of the rat life span. Thus, 2-, 4-, and 8-week vibrations in rats are equivalent to approximately 1, 2, and 4.5 years of vibration in humans, respectively. The preliminary findings from our pilot study at these different vibration time points revealed clear differences and showed that a process was continuing. Eight-week WBV, which is equivalent to approximately 4.5 years for most occupational drivers, is not a very long period; however, even at this point vibration-induced brain damage was substantial and prominent. (Fig 6). The physiological and pathological changes at the 2-, 4-, and 8-week time points have shown that WBV injury is a gradually accumulative and worsening process.

The dynamic development of accumulated injury from WBV is complex and has multiple causes. Major causes include1 mechanical vibrated force, such as repeated stretching of and impact upon tissues and cells2; physiological damage such as vibration-induced early vascular spasm; late vascular constriction causes brain ischemia damage3; anion damage, day–vibration and night-rest, induces vascular dilated reperfusion lesion by superoxide anions (O2−)4; biochemical damage, in which cellular membrane and associated membrane protein enzymes such as Ca-ATPase, Na/Ca2+ exchange enzyme disintegrate, which causes excessive calcium accumulation in the neural system. Calcium further activates multiple toxic enzymes followed by cascade injury and more severe cell damage. These degrees of injury are vibration time dependent and reveal a gradually worsening process. For example, in a medium size artery, MCA spasm had started at 2 weeks; obvious structural injury, endothelial cell loss (Fig 4, B), and brain capillary constriction (Fig 5) were seen at 4 weeks; the whole artery wall was broken at 8-weeks (Fig 4, C). The change affecting brain neurons was not seen in the 2-week study; mild surrounding brain-cell edema started in the 4-week WBV; obvious brain neuron atrophy and DSN appeared by 8 weeks (Fig 6).

WBV injury has its specific features. The mammalian brain has a firm skull that protects it from any outside striking force. However, the injury to the brain induced by WBV is transferred to the brain tissues from the vibrated whole body. In other words, the injury force is from inside, stretching, impacting, twisting, and shearing induced by the vibration waves. This inside force makes WBV-induced brain injury very insidious or invisible. Although brain injury is minimal in the early stage, which does not induce an immediate structural injury, it has the potential to accumulate over time when subjects are exposed to WBV for long periods, which was identified in the present study. The insidious nature of the WBV injury, the process, and its sequelae should be seriously regarded. Acute (short time) WBV causes cerebral vascular spasm, then constriction, decreased cerebral blood flow, and increased free radicals, thereby impairing brain function including judgment and reactive capability, which may be an important factor in MVAs. When the correlation analyses between behavior (maze time) and brain blood flow volume (via carotid artery), and between maze error numbers and vasoconstriction ratio (EC/EM) were performed, each coefficient of correlation (r) was greater than .62 (Fig 9), and all correlations were statistically significant (all P < .05 or less). Drivers usually feel fatigued and drowsy during long hours of driving. Our data suggest this so-called “fatigue” may actually be brain dysfunction resulting from WBV-induced injury.

Figure 9.

Figure 9

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Correlation graphs between behavior and blood supply to the brain. The graphs were from the 4-week study groups. (A) Correlation between the maze time and the blood flow volume of the carotid artery (−.62, P < .05). (B) Correlation between the maze error numbers and the vasoconstriction ratio of MCA (−. 80, P < .005). Abbreviations: CBF, carotid blood flow; EC/EM, vasoconstriction ratio, endothelial circumference (EC) divided by elastic membrane (EM). The lower the vasoconstriction ratio, the smaller the lumen is; MCA, middle cerebral artery; ME, maze error; MT, maze time.

Neural system lesions from MV-WBV have been discovered and confirmed by pathological evidence in our study, which has not been previously described. The exact biological mechanism of WBV inducing vascular and neural damage is still unknown. Locally, vibration force stimulates sympathetic nerve fibers to release norepinephrine and increases the smooth muscle sensitivity to norepinephrine.37 The smooth muscle spasm may be caused by direct stimulation from the vibration shearing force. The force causes endothelium and smooth muscle bulging into the lumen.38 Vacuoles form in the cytoplasm of the squeezed endothelial cells, and then the cells peel off from the underlying smooth muscle layer.

The damage to neurons and peripheral nerves results from direct shearing force and ischemia. A repeated vibrating–resting (day–night) condition is similar to ischemia–reperfusion. In addition, ischemia and the shear stress trigger nicotinamide adenine dinucleotide phosphate and xanthine oxidase to generate more O2−, which damages the neural system.17,39 Vibration–stretching and impacting force also impairs axoplasmic transportation, as shown by our previous study.40

The present study has shown that WBV injury is a cumulative process. The comparisons of the results among 2-, 4-, and 8-week vibrations showed obvious differences, and all were statistically significant (Tables 1, 2). In the 8-week vibration group, morphological lesions, such as thickened cerebral artery and capillary walls, may induce other secondary diseases, such as chronic hypertension41,42 or cerebrovascular diseases.43 According to Djindjić et al.’s41 cross-sectional study of 250 long-distance truck drivers, 45.2% were diagnosed with hypertension, and there was a high prevalence of cardiovascular risk factors. In our study, we also validated DSN increase and neuronal atrophy with long-term WBV; these changes could produce permanent impairment of memory, judgment, and reactive capabilities, and could potentially contribute to other neurological degenerative states. A simulated long-term further study, using time points such as 12 and 24 weeks, should be considered to validate chronic, permanent lesions in rats from WBV and to investigate preventive approaches.

We have shown that 4F restores the normal physiological balance of several mechanisms to ameliorate the effects of WBV. We selected and tested 4F for its preventive effects from WBV because 4F has special pharmacological properties. In some cases of the present study, it improves the results over the sham controls. We believe the following mechanisms to be part of the 4F preventive effects. First, 4F reduces WBV caused vascular spasm, endothelium injury, and ischemia–reperfusion that produce superoxide. Superoxide combines with NO to form peroxynitrite, which reduces NO bioavailability for vasodilatation and inhibits eNOS synthesis.44 This creates a positive feedback loop, increasing damage. Second, 4F actions are reported to include reduced apoptosis of endothelial cells, reduction of transforming growth factor beta (TGFβ) levels, absorption and removal of oxidized phospholipids, as well as restoration of heat shock protein 90 activation of eNOS.45 In endothelial cells, 4F restores a safe balance of NO and O2− generation.46 All these combine to prevent endothelial cell damage. Third, cortex neuronal injury may also be prevented by 4F because vibration produces vasoconstriction, which reduces blood flow to neurons via microcirculation. This increases secondary edema and inflammation, which eventually causes neuron necrosis, shown as DSNs. While most neurons are still functioning, they have become atrophied, which is shown as a reduced size. 4F prevents these harmful effects. Furthermore, several trials suggest that 4F increases high-density lipoprotein (HDL) to improve the recovery from vascular lesions,47–49 and to improve the recovery of the injured axonal myelin. A recent study further showed that 4F reduced systemic inflammation by modulating intestinal oxidized lipid metabolism.25 Also, 4F has been used in a recent clinical trial titled: “4-EVER: a Trial Investigating the Safety of 4F Endovascular Treatment of Infra-Inguinal Arterial Stenotic Disease” (ClinicalTrials.gov Identifier: NCT01413139), which was completed in September 2013. According to the authors, it has shown positive results.26

In summary, 4F has 5 major preventive effects for WBV injury: (1) increase in vascular dilation and decrease in endothelial injury; (2) increase in the concentration of blood NO and decrease in superoxide to prevent neuronal damage; (3) increase in HDL, which improves recovery of the endothelium; (4) protection of nerve fiber myelin, in our observation; this may be due to an increase in HDL; and (5) reduction in secondary inflammation, edema, and intraneural pressure, thereby preventing neuron atrophy and apoptosis. Taken together, this report indicates that 4F is a potential dynamic preventive medication for WBV injury. Conditioning using a single medication to prevent vibration injury is a new method, which has never been investigated before. As stated previously, travel by MVs, trains, tanks, helicopters, aircraft, or farm equipment induces WBV. After a short vibration time, vasoconstriction will reduce tissue blood flow, such as in the brain, therefore reducing fitness and increasing fatigue and weakness, and reducing judgment and reactive capabilities. Reducing these harmful effects by conditioning with 4F would also reduce accidents. A reduction of acute vibrational effects can also prevent chronic vascular and nerve injuries.

Acknowledgments

This research and development project was made possible by a research grant awarded and administered by the U.S. Army Medical Research and Materiel Command and the Telemedicine & Advanced Technology Research Center at Fort Detrick, MD (Grant number W81XWH-11-1-0694).

Preliminary portions of the present study have been published elsewhere (J Neurosci Res 2014 Dec 29. doi: 10.1002/jnr.23536.). Wiley Publishing has graciously permitted selected text and several figures from that article to be used in the current submission, which is expanded to include new data of the 8-week and 4F prevention studies.

Abbreviations

4F

human apolipoprotein A-I molecule mimetic

DDSA

dodecenyl succinic anhydride

DSN

dark shrunken neuron

EC

endothelial circumference

EC/EM

vasodilation degree

EM

elastic membrane

HDL

high-density lipoprotein

Hz

Hertz

LM

light microscopic study

MCA

middle cerebral artery

MV

motor vehicle

NCV

nerve conduction velocity

RIU

relative intensity unit

TEM

transmission electron microscopic

WBV

whole-body vibration

Footnotes

The authors have no competing financial interests to declare.

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