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Published online before print December 29, 2003, 10.1148/radiol.2302020417
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(Radiology 2004;230:377-382.)
© RSNA, 2003

Experimental Studies

In-Stent Restenosis Limitation with Stent-based Controlled-Release Nitric Oxide: Initial Results in Rabbits1

Young S. Do, MD2, Edward Y. Kao, MD, Fumikiyo Ganaha, MD, Hiroki Minamiguchi, MD, Koji Sugimoto, MD, Jane Lee, BS, Christopher J. Elkins, PhD, Philippe G. Amabile, MD, Michael D. Kuo, MD, David S. Wang, MD, Jacob M. Waugh, MD and Michael D. Dake, MD

1 From the Department of Cardiovascular and Interventional Radiology, Stanford University School of Medicine, 300 Pasteur Dr, Rm H-3647, Stanford, CA 94305. Received April 11, 2002; revision requested July 9; final revision received August 26, 2003; accepted August 27. Address correspondence to M.D.D. (e-mail: mddake@stanford.edu).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
PURPOSE: To evaluate effect of controlled stent-based release of an NO donor to limit in-stent restenosis in rabbits.

MATERIALS AND METHODS: Bioerodable microspheres containing NO donor or biodegradable polymer (polylactide-co-glycolide–polyethylene glycol) were prepared and loaded in channeled stents. Daily concentrations of NO release from NO-containing microspheres were assayed in vitro. NO- and polymer-containing (control) microsphere-loaded stents were deployed in aortas of New Zealand white rabbits (n = 8). Aortas with stents were harvested at 7 (n = 5) and 28 days (n = 3) and evaluated for cyclic guanosine monophosphate (cGMP) levels (7 days), number of proliferating cell nuclear antigen–positive cells (7 days), and intima-to-media ratio (7 and 28 days), with statistical significance evaluated by using one-way analysis of variance.

RESULTS: NO-containing microspheres released NO with an initial bolus in the 1st week, followed by sustained release for the remaining 3 weeks. Significant increase in cGMP levels and decrease in proliferating cell nuclear antigen–positive cells were found at 7 days for the NO-treated group relative to controls (P < .05). Intima-to-media ratio in the NO-treated group was reduced by 46% and 32% relative to controls at 7 and 28 days, respectively (mean, 0.14 ± 0.01 [standard error] vs 0.26 ± 0.02 at 7 days, P < .01; 1.34 ± 0.05 vs 1.98 ± 0.08 at 28 days, P < .01).

CONCLUSION: Stent-based controlled release of NO donor significantly reduces in-stent restenosis and is associated with increase in vascular cGMP and suppression of proliferation.

© RSNA, 2003

Index terms: Animals • Arteries, femoral, 92.1268, 92.411 • Arteries, restenosis, 92.121, 92.411 • Experimental study • Stents and prostheses


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Angioplasty with stent deployment is the most commonly used primary intervention for advanced cardiovascular occlusive disease. However, long-term effectiveness of this treatment remains limited by accelerated in-stent restenosis (1–3). Arterial restenosis results from a complex wound healing process that is affected by many factors; occurs in response to vessel injury; and is characterized by neointimal hyperplasia, elastic recoil, mural thrombus, and negative vascular remodeling (4–6). Stent implantation preserves luminal diameter in the near term by preventing early elastic recoil and some components of late vascular remodeling after angioplasty (7). However, long-term success remains limited because of the occurrence of in-stent restenosis, which is characterized primarily by neointimal formation. In-stent neointima arises from the accumulation of vascular smooth muscle cells (VSMCs) and the deposition of extracellular matrix (2,3,8).

Recently, a great deal of attention has been focused on the potential of drug-eluting stents to limit the critical pathologic processes that contribute to neointimal formation and late in-stent restenosis. Local drug delivery offers the potential to achieve high local concentrations with low systemic effects. Such delivery can be achieved by means of the incorporation of active agents into biodegradable polymers to be used as a reservoir for sustained local drug delivery (9–13). Preliminary results from noncontrolled commercial trials with simple polymer-coated stent release of agents such as paclitaxel or sirolimus (formerly known as rapamycin) show improvements in early restenosis rates (14,15). However, as with brachytherapy, the long-term effects of these stents may prove quite different, particularly after complete elution of therapeutic agents. Furthermore, these agents simply reduce cellular gains in neointima rather than regulate the underlying physiologic processes responsible for neointimal formation. Improved stent-mediated delivery technology and the use of physiologic agents to regulate the processes that contribute to restenosis offer promise for both better understanding and improved management of accelerated in-stent plaque progression. For this reason, we have developed a stent-based controlled drug-delivery platform that does not itself alter plaque progression or local inflammation (16).

Among the many physiologic agents that have been proposed to regulate accelerated restenosis, NO may prove particularly effective. NO, formed from L-arginine by means of NO synthase, induces cyclic guanosine monophosphate (cGMP)–dependent vasorelaxation and inhibits platelet adhesion and aggregation, VSMC proliferation and intimal migration, and extracellular matrix formation (17–21). Since each of these processes plays a key role in neointimal formation and accelerated restenosis, NO represents a potentially important physiologic anti–neointimal formation agent. In animal models, enhanced vascular NO production has been shown to inhibit restenosis after simple vascular injuries (22–26).

We hypothesize that controlled release of an NO donor from a channeled stent can mediate NO-dependent effects to limit VSMC proliferation and pathologic neointimal formation after injury. Thus, the purpose of our study was to evaluate the effect of controlled stent-based release of NO donor to limit in-stent restenosis in a rabbit model.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
Stent Design
The stent design was custom fabricated from a basic coronary stent (Palmaz-Schatz; Johnson & Johnson, Warren, NJ). The unexpanded stents are 1.6 mm in diameter and 10 mm in length, with 10 longitudinal struts evenly spaced circumferentially. This stainless steel stent is designed to be balloon expandable up to a 5-mm diameter. The basic slotted-tube design was modified by means of electrodischarge machining (EDM; Norman-Noble, Cleveland, Ohio) channels 80 µm wide by 60 µm deep along the length of the abluminal (ie, toward the wall) outer surface of each strut. The channels were machined from end to end in order to allow drug delivery both to the wall and to the tissue surrounding the stent end. Biodegradable microspheres, which store and release therapeutic agents, were mechanically loaded into these channels and covalently anchored as previously described (16).

Microsphere Preparation
Biodegradable poly(lactic-co-glycolic acid)–polyethylene glycol microspheres were prepared as a modification of previously described techniques (22,25,27). A mixture of 75:25 poly(lactic-co-glycolic acid) (Polysciences, Warrington, Pa) and polyethylene glycol 8000 (Fisher Scientific, Fair Lawn, NJ) in a ratio of 8:1 was used with the double-emulsion technique to generate microspheres with a final diameter of 10 µm and a degradation time of approximately 4 weeks. Additionally, a buffer with pH 7.4 was incorporated to limit local pH changes in order to stabilize incorporated drugs and render the microspheres more biocompatible (28). During the microsphere manufacturing process, the NO donor to be delivered was added as an aqueous solution to the polymer solution to make NO microspheres. The NO-nucleophile complex in this study, N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)ethanamine (NOC-12; Calbiochem, San Diego, Calif), releases free NO in a pH-dependent fashion. With appropriate choice of nucleophile, the NO-nucleophile complex can release NO rapidly (during 1–2 minutes) or slowly (during several days). In this study, NO donor releases two equivalents of NO and has a half-life of 327 minutes once released into solution (26,29). Blank (polymer only) microspheres were prepared as controls.

Gel Preparation and Stent Loading
Stents were pretreated to form a metal oxide–methoxysilane-monomethacrylate link to the metal oxide layer of the stent surface. Stents were pretreated with 2% 3-methacryloxypropyltrimethoxysilane (Polysciences) in 75% ethanol and were heat cured for 60 minutes at 105°C. Stent channels were then manually loaded with control or NO-containing microspheres. Stents were temporarily ensheathed in a silicone tube, and gels (20% mixture of polyethylene glycol–dimethacrylate [Polysciences] and polyethylene glycol in a ratio of 3:2) were introduced to fill unoccupied spaces within the channels and were subsequently polymerized to form gel-stent links with the exposure to UV-A overnight. The silicone tubing was then withdrawn. Stents were loaded on 5.0-mm x 2.0-cm noncompliant angioplasty balloons (Jupiter; Cordis, Miami, Fla) and lyophilized overnight prior to use.

In Vitro NO Release
Although the buffered polymeric system used here should not interfere with drug stability, sustained release of NO from microspheres was confirmed directly. Control and NO-containing microspheres were assayed to confirm daily release of NO during a 4-week period. Daily release of NO at 37°C was collected by hydrating and incubating 5-mg doses of NO or control microspheres (n = 3) in a rotary bath with 1-mL volumes of phosphate-buffered saline. The volumes were collected and replaced with volumes of fresh phosphate-buffered saline daily for 28 consecutive days. To measure error in this assay, three parallel collections were obtained for both NO-containing and control microspheres. At the end of 28 days, a chromogenic assay for NO concentration was performed in all collected samples by using an NO assay kit (Calbiochem) and a microplate reader (ELx 808 Ultra Microplate Reader; Biotek Instruments; Winooski, Vt) at 540 nm. Comparison with standard samples of NO at known concentrations allowed absorbance data to be converted to concentrations of NO. From these data, release curves were obtained that could be compared at each time point to test for statistical significance. Since control microspheres would not generate positive signal, the results from controls define background levels for this assay.

In Vivo Stent Implantation
Adult male New Zealand white rabbits (Myrtles Rabbitry, Thompson Station, Tenn) that weighed 3.8–4.2 kg were used in accordance with National Institutes of Health and institutional guidelines (eight animals per group). While general anesthesia with isoflurane was induced through inhalation, an arteriotomy of the femoral artery was performed, and a 5-F introducing sheath was placed. With fluoroscopic guidance, sterilized channeled stents loaded with either control or NO-containing microspheres were deployed in the infrarenal abdominal aorta. Stents were dilated with a 5-mm angioplasty balloon at a balloon inflation pressure of 8 atm (810 kPa) to a final lumen size that was 125% of the baseline. Care was taken to ensure that no arterial branches were occluded by the stent segment. Pre- and postdeployment digital subtraction aortograms were obtained for each procedure. The rabbits were fed a 0.25% cholesterol diet after the intervention and were maintained on that diet until artery and stent harvest at 7 and 28 days. Procedures were performed by a group of interventionalists (Y.S.D., E.Y.K., F.G., H.M., K.S., M.D.K., D.S.W., J.M.W.).

Tissue Harvesting and Preparation
At 7 (five per group) or 28 (three per group) days after stent deployment, the animals underwent total-body perfusion-fixation with 2 L of 2% neutral buffered formalin after 2 L of normal saline was infused. This procedure was performed by a group of several authors. The stent-containing arterial segments were then immediately harvested. The 7-day aortic specimens were incised longitudinally, and stents were removed. The harvested aortic specimens were divided into two equal segments. One segment was fixed in 10% neutral buffered formalin prior to being embedded in paraffin for light microscopic and morphologic analysis. The remaining segment was snap frozen for subsequent cGMP assay. The 28-day aortic specimens were harvested and fixed, with stents in situ, in 10% neutral buffered formalin and were embedded in a medium (PolyBed; Polysciences, Warrington, Pa) for light microscopic and morphologic analysis.

Vessel cGMP Assay
To confirm the delivery and diffusion of stent-based release of NO into the vessel wall, cGMP levels in the vessel were measured from the snap-frozen 7-day aortic specimens from both groups with control and NO-containing microspheres (n = 5). All frozen segments were treated as previously described (26). cGMP levels from the samples were assayed by using a commercially available immunoassay kit (Amersham Life Science, Arlington, Ill). Results were expressed as picomoles of cGMP per milligram of trichloroacetic acid–precipitatable protein.

Evaluation of Neointima: Intima-to-Media Ratios
Three serial (5-µm-thick) cross sections were obtained from the distal face of each 7-day arterial segment (15 sections per group). Double staining with elastica van Gieson–Masson trichrome or elastica van Gieson–hematoxylin stains was performed on each section. Five serial cross sections were obtained from the distal end of each 28-day specimen (15 sections per group). These sections were grossly stained by using modified Verhoeff elastic staining that consisted of Verhoeff staining for 2 hours and van Gieson staining for 3 hours. High-spatial-resolution digital images of the stained 7- and 28-day specimens were obtained at x100 magnification by using a camera (SPOT true-color; Diagnostic Instruments, Silver Spring, Md) with a microscope (Nikon E600 with Plan Apochromat Lenses; Nikon, Melville, NY). The 28-day specimens were evaluated for thrombosis and neointimal formation. With software (Image Pro Plus; Media Cybernetics, Silver Spring, Md), the cross-sectional area of the intima and the media was determined by a blinded observer (F.G.), and the ratio of intimal area to medial area was tabulated for each specimen. Results were independently confirmed by another blinded observer (J.M.W.).

Evaluation of VSMC Proliferation
Proliferating cell nuclear antigen was measured to evaluate VSMC proliferation. Three sections per arterial segment from the 7-day aortic specimens (15 per group) were incubated overnight with a primary monoclonal antibody to proliferating cell nuclear antigen (Dako, Carpinteria, Calif) at a dilution of 1:20. After the sections were washed in phosphate-buffered saline, they were then incubated with an alkaline phosphate–conjugated secondary antibody to mouse immunoglobulin (Calbiochem) at a dilution of 1:100. A nitro-blue tetrazolium/5-bromo-4-chloro-3-indolyphosphate p-toluidine substrate (Pierce, Rockford, Ill) was then used as a chromogen. A blinded observer (E.Y.K.) evaluated the presence and distribution of proliferating cell nuclear antigen–positive nuclei by using thresholding with the previously mentioned software after red-channel extraction. Automated results were manually confirmed for each by one blinded observer and independently by the other blinded observer.

Statistical Analysis
All data were expressed as mean ± standard error. For all experiments, mean and standard error were determined for each group, and statistical comparisons were made by using one-factor analysis of variance for repeated measures, with significance evaluated at the 95% level. In addition, Bonferroni, Tukey-A, and Student–Newman-Keuls post hoc testing was performed with software (Statview, SAS Institute, Cary, NC; SPSS 6.1 for Macintosh, Prentice Hall, Upper Saddle River, NJ). Individual P values were determined and are reported.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 
In Vitro NO Release
Release curves generated from the in vitro functional assay of NO and control microspheres are shown in Figure 1. The overall pattern of NO release from NO-containing microspheres was that of an initial bolus in the 1st week, followed by sustained release during the next 3 weeks. As expected, NO-containing microspheres released significantly more NO compared with controls at all time points (P < .01).



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  		Figure 1. Graph shows mean daily in vitro release of NO according to week for control and NO-containing microspheres. Values represent mean ± standard error. Error bars = standard error, * = P < .01.

 
In Vivo Stent-based NO Release
Vascular cGMP levels for groups with NO-containing microspheres relative to controls were examined at 7 days, and results are summarized in Figure 2. Vascular cGMP levels were significantly increased in the group with NO-containing microspheres (17.66 ± 4.00) relative to controls (7.69 ± 1.45, P < .01). These results demonstrate successful stent-based release of functional NO and sufficient NO diffusion to the vascular wall to allow VSMC conversion of guanosine triphosphate to cGMP.



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  		Figure 2. Graph shows in vitro vascular cGMP levels. Vascular cGMP levels at 7 days after in vivo stent deployment and delivery of control and NO-containing microspheres. Values represent mean ± standard error. Error bars = standard error, * = P < .01.

 
Proliferating Cell Nuclear Antigen Immunohistochemical Analysis
The effect of stent-based release of NO on VSMC proliferation was subsequently examined relative to controls at 7 days (n = 5), and results are summarized in Figure 3. Stent-based release of NO resulted in a significant decrease in proliferating cell nuclear antigen–positive cells per section (15.67 ± 1.07) relative to that of controls (25.07 ± 2.65, P < .01).



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  		Figure 3. Graph shows VSMC proliferation after stent implantation, with total proliferating cell nuclear antigen (PCNA)-positive cells per cross section at 7 days after delivery of control and NO-containing microspheres. Values represent mean ± standard error. Error bars = standard error, * = P < .01.

 
Neointimal Formation
The effect of stent-based NO release on in-stent neointimal formation was evaluated relative to that of controls at both 7 (n = 5) and 28 (n = 3) days, and results are summarized in Figure 4. At 7 days, the intima-to-media ratio of NO-treated groups (0.14 ± 0.01) was reduced by 46% relative to that in controls (0.26 ± 0.02, P < .01). At 28 days, the intima-to-media ratio was reduced by 32% in NO-treated groups (1.34 ± 0.05) relative to that in controls (1.98 ± 0.08, P < .01). Treatment with NO thus resulted in a significant reduction of in-stent neointimal formation relative to that in controls at both 7 and 28 days. There was no evidence of luminal thrombi in either group at 28 days. Representative photomicrographs obtained at 28 days are presented in Figure 5.



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  		Figure 4a. Graphs show intima-to-media (area) ratio at (a) 7 days and (b) 28 days after in vivo stent deployment and delivery of control and NO-containing microspheres. Values represent mean ± standard error. Error bars = standard error, * = P < .01.

 


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  		Figure 4b. Graphs show intima-to-media (area) ratio at (a) 7 days and (b) 28 days after in vivo stent deployment and delivery of control and NO-containing microspheres. Values represent mean ± standard error. Error bars = standard error, * = P < .01.

 


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  		Figure 5a. (a, b) Anteroposterior abdominal aortograms of preharvest arteries at 28 days with (a) control stents or (b) stents loaded with NO-containing microspheres. Arrows denote proximal and distal margins of stent. No arterial perforations or complications are evident in either group. (c, d) Postharvest arterial cross sections obtained 28 days after treatment with (c) control stents or (d) stents loaded with NO-containing microspheres. Degree and composition of neointima (N) relative to stent tines (S) and media (M) are depicted. Note exuberant neointimal formation in control relative to NO-treated specimen. (Verhoeff-van Gieson stain; original magnification, x200.)

 


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  		Figure 5b. (a, b) Anteroposterior abdominal aortograms of preharvest arteries at 28 days with (a) control stents or (b) stents loaded with NO-containing microspheres. Arrows denote proximal and distal margins of stent. No arterial perforations or complications are evident in either group. (c, d) Postharvest arterial cross sections obtained 28 days after treatment with (c) control stents or (d) stents loaded with NO-containing microspheres. Degree and composition of neointima (N) relative to stent tines (S) and media (M) are depicted. Note exuberant neointimal formation in control relative to NO-treated specimen. (Verhoeff-van Gieson stain; original magnification, x200.)

 


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  		Figure 5c. (a, b) Anteroposterior abdominal aortograms of preharvest arteries at 28 days with (a) control stents or (b) stents loaded with NO-containing microspheres. Arrows denote proximal and distal margins of stent. No arterial perforations or complications are evident in either group. (c, d) Postharvest arterial cross sections obtained 28 days after treatment with (c) control stents or (d) stents loaded with NO-containing microspheres. Degree and composition of neointima (N) relative to stent tines (S) and media (M) are depicted. Note exuberant neointimal formation in control relative to NO-treated specimen. (Verhoeff-van Gieson stain; original magnification, x200.)

 


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  		Figure 5d. (a, b) Anteroposterior abdominal aortograms of preharvest arteries at 28 days with (a) control stents or (b) stents loaded with NO-containing microspheres. Arrows denote proximal and distal margins of stent. No arterial perforations or complications are evident in either group. (c, d) Postharvest arterial cross sections obtained 28 days after treatment with (c) control stents or (d) stents loaded with NO-containing microspheres. Degree and composition of neointima (N) relative to stent tines (S) and media (M) are depicted. Note exuberant neointimal formation in control relative to NO-treated specimen. (Verhoeff-van Gieson stain; original magnification, x200.)

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
 
In order to evaluate the effect of NO release on neointimal hyperplasia after stent implantation, we prepared bioerodable polymer microspheres for releasing an NO donor. In this study, a particular NO-nucleophile complex was used as an NO donor (29). This NO-nucleophile complex exhibits potent vasorelaxant (30), antiplatelet (31), and antimitogenic effects (32) in vitro and in animal models. Certain polymers themselves can be responsible for local inflammation and neointimal formation primarily because of their alterations of pH. However, buffered polymer systems such as those used here prevent these effects (33–35). We have previously validated that the polymers used here do not perturb plaque formation or enhance local inflammation (16). To confirm that the preparation method did not destabilize the NO donor, we evaluated release of NO generation in vitro from poly(lactic-co-glycolic acid)–polyethylene glycol microspheres. These experiments revealed a bolus of NO generation followed by sustained NO release during 4 weeks, as predicted on the basis of the polymer system selected. Since the majority of physiologically relevant regulation of plaque progression after stent implantation occurs within the first 2–4 weeks, this time course allows characterization of end-point, as well as key, early pathophysiologic indicators.

NO is a simple lipophilic gas that easily diffuses through cell membranes and, thus, does not act through a membrane-bound receptor. NO exerts a number of diverse biologic actions through the activation of guanylate cyclase, which results in an increase in the second messenger cGMP. This increase in cGMP is responsible for the physiologic actions of NO (17,18). NO produced by endothelial cells is the major physiologic regulator of vessel tone (17,18) and inhibits platelet aggregation and adhesion to endothelium (19,36,37).

Since NO actions are mediated through cGMP, we used an assay of normalized cGMP levels to confirm stent-based NO release in vivo. This assay confirms not only that NO is being released from the stent platform but also that NO diffuses into the wall at levels sufficient to provide marked activation of the appropriate second-messenger system.

Since there is also ample evidence that NO directly inhibits VSMC proliferation and migration (21,35), which are largely responsible for cellular gains in neointima, we evaluated the effect of controlled stent-based NO release on VSMC proliferation in vivo. Results of this experiment confirm that stent-based NO release significantly limits proliferation of VSMCs after intraaortic stent implantation. These results are consistent with the prior observation that immediate single-dose supplementation of NO in vein grafts by using an NO donor results in significant decreases in intimal hyperplasia (38). However, results of other studies have shown that the antiproliferative effect of NO donors on cultured VSMCs is greatest when a given molar quantity is delivered slowly throughout the observation period (32). This may be in part because maximal DNA synthesis in the neointima and the media occurs within 7 days and lasts through at least 14 days after vascular injury (39). Thus, continuous long-term local release of NO from a nonperturbing polymer-based system, as in this work, may offer substantial advantages for inhibition of VSMC proliferation. Here, we find that local stent-based release of an NO donor proves sufficient to limit neointimal formation after arterial injury.

Various strategies, which include gene therapy (26), brachytherapy (40), perivascular drug delivery, (23,28) or stent-mediated drug delivery (12,13), have been suggested to reduce in-stent restenosis. Comparison of our results with those of other strategies is difficult, given the differences in approach. However, stent-based controlled release of anti– neointimal formation agents becomes particularly appealing because of the local nature of in-stent restenosis, the dramatic increases in stent use, and the observation that the major part of in-stent restenosis results from neointimal hyperplasia (7).

Practical application: We report here that controlled stent-based delivery of NO can limit VSMC proliferation and reduce subsequent in-stent restenosis. This method of long-term local NO delivery during stent implantation may offer the advantage of preservation of desired regional effects while systemic effects such as hypotension are minimized. Refinements of this strategy in terms of stent design, dosing optimization, and expansion of subject numbers will ultimately be required before this platform could offer substantial clinical benefits for patients receiving stents. Furthermore, longer time points, together with additional work, must be examined to confirm a lack of species specificity in dosing. Direct comparison with commercial drug-delivery stents may also provide future insights.


   FOOTNOTES
 
2 Current address: Department of Radiology, Samsung Medical Center, Sungkyunkwan University College of Medicine, Seoul, Korea. Back

Abbreviations: cGMP = cyclic guanosine monophosphate, VSMC = vascular smooth muscle cell

Author contributions: Guarantor of integrity of entire study, M.D.D.; study concepts, Y.S.D., C.J.E., J.M.W., M.D.D.; study design, Y.S.D., C.J.E., M.D.K., J.M.W., M.D.D.; literature research, Y.S.D., F.G., C.J.E., J.M.W.; experimental studies, Y.S.D., E.Y.K., F.G., H.M., K.S., C.J.E., P.G.A., J.M.W.; data acquisition, Y.S.D., E.Y.K., F.G., H.M., K.S., J.L., P.G.A., D.S.W., J.M.W.; data analysis/interpretation, Y.S.D., E.Y.K., F.G., H.M., K.S., J.L., P.G.A., M.D.K., D.S.W., J.M.W., M.D.D.; statistical analysis, D.S.W., J.M.W., M.D.D.; manuscript preparation, Y.S.D., E.Y.K., J.L., J.M.W., M.D.D.; manuscript definition of intellectual content, J.M.W., M.D.D.; manuscript editing, E.Y.K., J.L., C.J.E., P.G.A., J.M.W., M.D.D.; manuscript revision/review, E.Y.K., J.L., M.D.K., J.M.W., M.D.D.; manuscript final version approval, all authors


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
DISCUSSION
 REFERENCES
 

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