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Inhibition of Inducible Nitric Oxide Synthase Promotes Recovery of Motor Function in Rats After Sciatic Nerve Ischemia and Reperfusion

Posted on: Friday, 5 August 2005, 09:00 CDT

Purpose: To investigate the effects of inhibition of inducible nitric oxide synthase (iNOS) on the recovery of motor function in the rat sciatic nerve after ischemia and reperfusion injury.

Methods: A 10-mm segment of the sciatic nerve from 169 rats had 2 hours of ischemia followed by up to 42 days of reperfusion. The animals were divided into 2 groups that received either iNOS inhibitor 1400W or the same volume of sterile water subcutaneously. A walking track test was used to evaluate the motor functional recovery during reperfusion. Statistical analysis was performed for the measurements of the sciatic functional index (SFI) by using 2- way analysis of variance; 1-way analysis of variance was used for the post hoc analysis of specific values at each time point of the SFI measurement.

Results: 1400W-treated rats had earlier motor functional recovery than controls, with a significantly improved SFI between days 11 and 28. Histology showed less axonal degeneration and earlier regeneration of nerve fibers in the 1400W group than in the controls. Inducible NOS messenger RNA and protein were up-regulated during the first 3 days of reperfusion but there was a down- regulation of neuronal NOS and up-regulation of endothelial NOS in control animals. 1400W treatment attenuated the increase of iNOS but had no effect on neuronal NOS and endothelial NOS.

Conclusions: Our results indicate that early inhibition of iNOS appears to be critical for reducing or preventing ischemia and reperfusion injury. (J Hand Surg 2005;30A:826-835. Copyright 20052005 by the American Society for Surgery of the Hand.)

Key words: 1400W, walking track testing, axonal regeneration, peripheral nerve, rat.

Ischemia and reperfusion (I/R) injury leads to complex pathophysiologic changes that compromise tissue function and cause an irreversible lack of tissue perfusion in many organs.1-3 Compared with essential organs such as the brain and heart in which ischemia may cause fatal effects, less attention has been directed toward I/ R injury in the peripheral nervous system (PNS). Peripheral nervous system injury resulting from I/R, however, is still a common clinical problem associated with acute trauma (eg, replantation, transplantation, tourniquet injury, compartment syndrome, Saturday night palsy) and chronic conditions (carpal tunnel syndrome, tumors, callus, etc). This injury poses a challenge because the degree of injury and time course of recovery vary considerably. Researchers have recognized that multiple factors including leukocytes, cytokines, adhesion molecules, nitric oxide (NO), and cyclooxygenase contribute to PNS I/R injury.4-6

Nitric oxide is a messenger molecule that is involved in many physiologic functions.2,7-9 The PNS functions regulated by NO include neurotransmission,10 blood flow,11 and nonvascular smooth muscle relaxation12 among others.13 Nitric oxide is synthesized from L-arginine by NO synthase (NOS).9 All 3 major NOS isoenzymes have been found in the nervous system.10,14 Neuronal NOS (nNOS) is expressed in the spinal cord,15 dorsal root ganglia,16 axons, Schwann cells,14 and end plates.17 Endothelial NOS (eNOS), in addition to expression in endothelial cells, also is expressed in the central nervous system.18 Inducible NOS (iNOS) is induced by endotoxins and cytokines and produces large amounts of NO to exert a defense function and is found in numerous cell types.19 When NO release is excessive iNOS causes marked cellular toxicity.20

Nitric oxide also is involved in multiple pathologic events in many organs and diseases. Up-regulated iNOS has been implicated in I/ R-associated pathology in skeletal muscle,21 cultured myocytes,22 myocardial infarction,23 and isolated heart,24 brain,25,26 liver,27 kidney,28 and pancreas tissues.29 We have shown that inhibition of iNOS significantly improves microcirculation and contractile function in I/R skeletal muscle.30-32 Inducible NOS-knockout mice show reduced I/R injury in the kidney,33 skeletal muscle,34,35 and brain.36 The exact role of iNOS in I/R injury still is controversial, however,37 and contradictory data also have been reported.38 In the PNS we have shown that I/R regulates NOS in rat sciatic nerve, suggesting a potential role for NO in PNS I/R injury.14 Among the 3 NOS isoforms iNOS up-regulation leads to excess NO production to exacerbate I/R injury. Our results are supported by findings from rat I/R sciatic nerve39 and focal ischemia of the central nervous system40 but differ from the results of studies of injured peripheral nerve in mice lacking iNOS.41,42 Thus the role of iNOS in PNS I/R injury needs further clarification.

This study was designed to examine the hypothesis that inhibition of iNOS will improve peripheral nerve recovery and muscle function. A highly selective iNOS inhibitor N- [3(aminomethyl)benzyl)acetamidine] (1400W) was administered, and the motor function and the levels of NOS messenger RNA (mRNA) and protein were measured in the reperfused rat sciatic nerve.

Materials and Methods

A total of 169 female Sprague-Dawley rats (weight, 175-200 g) were used to compare nerve motor function recovery with and without subcutaneous administration of 1400W. Control animals received the same volume of purified water.

Surgical Procedures

Rats received an intraperitoneal injection of anesthesia (Nembutal 50 mg/kg body weight; Abbott Laboratories, North Chicago, IL). The gluteal area was shaved and swabbed with Betadine solution (The Purdue Frederick Company, Norwalk, CT). Through a gluteal- splitting approach the right sciatic nerve was exposed and isolated from the surrounding soft tissue under an operating microscope. By using a specially designed compression device43 a 10-mm segment of the nerve proximal to the bifurcation of the sciatic nerve into the tibial and peroneal nerves was placed between a 10-mm-wide anvil, and a matching 10-mm-wide metal surface was mounted on the tip of the nonrotating micrometer spindle. The spindle was advanced until the desired 100-g load was achieved, as measured by an integral load cell. The load was maintained for 2 hours. This procedure has been shown to mimic an ischemic condition in the sciatic nerve.43,44 During the procedure the nerve was kept moist with 37C saline solution. After ischemia the proximal and distal ends of the ischemic segment were marked with 10-0 nylon sutures (Ethicon, Somerville, NJ) and the wound was closed with 4-0 silk running sutures. The rats were allowed to recover from the anesthesia and were handled in accordance with the guidelines of the Animal Care and Use Committee of the National Institutes of Health.

The study consisted of 2 phases: in phase I a total of 18 rats were used to evaluate the effects of the dose and duration of 1400W administration on motor functional recovery. Doses of 3 and 10 mg/ kg of 1400W administered during ischemia, 10 minutes before reperfusion, were compared (3 rats for each group). In addition the effects of longer administration durations of the 3 mg/kg dose of 1400W of 1, 3, 7, and 14 days on motor functional recovery also were compared (3 rats for each duration). A total of 3 mg/kg of 1400W was administered every 12 hours after the initial administration until the end of the administration period. In phase II the optimal 1400W dose and administration duration determined in phase I or the same volume of sterile purified water was administered to the remaining 151 animals.

Table 1. Oligonutleotides of Primers for PCR

Assessment of Motor Function

By using a walking track test the motor function of the rats was evaluated at days 1, 7, 11, 14, 18, 21, 25, 28, 35, and 42 of reperfusion (19 rats for each group). This procedure has been described previously.43,44 Briefly the rats were allowed to walk down a corridor with a dark shelter at the end. An 8 42-cm piece of photocopying paper impregnated with a 0.5% solution of the anhydrous form of bromphenol blue (Sigma Chemical, St. Louis, MO) in absolute acetone was placed on the floor of the walking corridor. The hind paws were moistened on a water-soaked sponge and the rats were allowed to walk down the corridor, leaving blue footprints on the paper. The distance between the first and fifth toes (TS), the distance between the second and fourth toes (IT), and the print length (PL) were measured. The sciatic functional index (SFI) was calculated according to the following equation.45

In the formula E refers to the experimental hind limb and N refers to the opposite normal hind limb. A total of O indicates normal nerve function and -100 represents complete dysfunction.

Quantitative Real-Time ReverseTranscription Polymerase Chain Reaction

Twelve I/R nerve segments and the opposite non-I/R normal nerve segments (as a normal control) from each group were harvested at 3 hours and at 1, 3, and 7 days after ischemia; placed in solution (RNA later; Ambion, Austin, TX) to stabilize and protect RNA in fresh samples; incubated at 4C overnight; and then stored at -20C. The harvested samples were pooled, 4 nerve segments for each. Total RNA from each pooled sample was extracted (RNeasy Mini kit plus DNase I digestion; Qiagen, Valencia, CA) and quantitated (RiboGreen; Molecular Probes Inc., Eugene, OR). Reverse transcription was performed ina total volume of 20 L containing 0.4 g of total RNA. First-strand complementary DNA was synthesized (Superscript II Reverse Transcriptase kit; Invitrogen, Carlsbad, CA) and quantified (PicoGreen; Molecular Probes, Inc.). Quantitative real-time polymerase chain reaction (PCR) was performed (iCycler Thermal Cycler and iQ SYBR Green Supermix real-time PCR kit; Bio-Rad, Hercules, CA). The primer sequences and product size for each NOS and 18S ribosomal RNA (about 2000 nucleotides long, as an internal control) are summarized in Table 1. The relative amounts of the target genes were calculated by the comparative threshold cycle method.14,30

Western Blot Analysis

Twelve I/R nerve segments and the opposite non-I/R normal nerve segments (as a normal control) from each group were harvested at 3 hours and at 1, 3, 7, 14, 21, and 42 days after ischemia, frozen in liquid nitrogen immediately, and then stored at -8O0C. The harvested samples were pooled, 2 nerve segments for each, and were homogenized in boiling lysis buffer (1% sodium dodecyl sulfate, 1.0 mmol/L sodium orthovanadate, 10 mmol/L Tris pH7.4) and microwaved for 10 to 15 seconds. The insoluble material was discarded after centrifugation of the homogenate according to the manufacturer's instructions (BD Biosciences, San Diego, CA). Protein concentrations were determined (BCA kit; Pierce, Rockford, IL). Proteins (20 μ%) were loaded onto NuPAGE 3%-8% Tris-acetate gel (Invitrogen, Carlsbad, CA) and then transferred onto a polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat dry milk in tri- buffered saline at room temperature for 1 hour, followed by incubation with monoclonal primary antibody for nNOS (1:1500; BD Transduction Laboratories, Lexington KY), eNOS (1:750; BD Transduction Laboratories), and polyclonal primary antibody for iNOS (1:750; Upstate Cell Signaling Solutions, Lake Placid, NY) at 4C overnight. After washing in TBS-T 3 times the blot was incubated with 1:5000 horseradish-peroxidase-labeled goat anti-mouse immunoglobulin G for 1 hour at room temperature for both nNOS and eNOS. For iNOS detection the blot was incubated with 1:12,000 horseradish-peroxidase-labeled goat anti-rabbit immunoglobulin G. After washing in TBS-T 3 times the immunoreactivity was detected (SuperSignal West Pico Chemiluminescent Substrate detection kit; Pierce). Photographic film (Kodak, Rochester, NY) was used to visualize chemiluminescent signals. Actin control was used to ensure equal loading.14,30

Histologic Evaluation

After the walking track test the ischemic segments of the sciatic nerve were harvested for histologic examination, 2 samples from each group, at days 7, 11,21, and 42. The samples were fixed on a thin wooden stick and stored in 4% glutaraldehyde for at least 24 hours at 4C. The nerve sample then was postfixed in 1% osmium tetroxide, dehydrated in increasing concentrations of ethanol, and embedded in Epon 812 (Structure Probe, Inc., West Chester, PA). sections were stained with toluidine blue.

Statistical analysis was performed for the measurements of the SFI by using 2-way analysis of variance and the values were represented as means standard error of the mean (SEM). Changes in mRNA and protein levels in I/R nerves were expressed as a percentage of the level of the opposite nonischemic normal nerves and the values were presented as means SD. A 1-way analysis of variance was used for the post hoc analysis of specific values at each time point of the SFI measurement and for the comparison of reverse- transcription PCR and Western blot results. A p value of less than .05 was considered statistically significant.

Results

Gross Observation During Reperfusion

Immediately after ischemia the compressed segments of all nerves were flattened slightly. Nerve continuity, however, was not interrupted grossly. At day 1 after ischemia all right limbs were paralyzed. All animals survived with no wound infection or selfmutilation at the time of nerve sample harvest.

Dose and Duration Responses of 1400W

The pilot study showed that all 1400W-treated groups had an earlier recovery of the SFI measurement than the water-treated control group between days 11 and 25. The time course of motor functional recovery was not significantly different between the lower dose (3 mg/kg 1400W) and higher dose (10 mg/kg). Similarly there was no significant difference in the SFI measurement between the durations of 1400W administration from the initial administration to consecutive administration for 14 days. Accordingly a dose of 3 mg/kg 1400W and a duration of 1 day were selected for administration in phase II of the study.

Figure 1. Time course of the SFI measurement after 2 hours of ischemia of rat sciatic nerve with 1400W or water administration. The error bars represent the SEM. Significant improvement of the SFI was found in 1400W-treated rats over water-treated rats between days 11 and 28. *p < .05; **p < .01; and ***p < .001 compared with the water group. [diamonds], Water; [black triangle up], 1400W.

Assessment of Motor Function

At day 1 after ischemia there were no measurable hind-paw prints from the ischmie limbs in both the experimental and control groups. The motor functional recovery started at day 11 in both groups, with an SFI of-90.5% 4.6% (mean SEM) in controls and of-82.2% 4.4% in the 1400W group. At this point and at all subsequent time points up to and including day 28 (13.3% 6.4% in controls,-7.8% 4.1% in the 1400W group) the 1400W group had a superior degree of functional recovery than controls, with a significantly improved SFI between days 11 and 28 (p < .05 to <.001) (Fig. 1). At day 35 motor function was recovered to near-normal levels in both groups.

Dynamics of NOS mRNA During Reperfusion

During reperfusion the expression of iNOS mRNA was up-regulated significantly from normal in controls at 3 hours, 24 hours, and 3 days of reperfusion, with a 43-, 13-, and 6-fold increase from normal, respectively. These values in the 1400W group were (p < .05 to <.01) attenuated significantly to 13-, 4-, and 3-fold increases, respectively (Fig. 2A). At day 7 the iNOS mRNA level decreased to close to normal, with no significant difference between the 2 groups.

Figure 2. The effects of 1400W on the expression of the 3 NOS mRNAs in rat sciatic nerve after 2 hours of ischemia and up to 7 days of reperfusion. The error bars represent the SEM. (A) iNOS mRNA, (B) nNOS mRNA, (C) eNOS mRNA. *p < .05 and **p < .01 compared with the water group. (A-C) [white square], Control; [black square], 1400W.

The nNOS mRNA level at 3 hours of reperfusion was reduced to 77% 33% (mean SD) and 51% 6% of normal in the control and 1400W groups, respectively. The nNOS levels further decreased to 61.4% 43% and 46.9% 19%, respectively, at day 1 and started to recover afterward (Fig. 2B). Although nNOS mRNA was lower in the 1400W group than in controls at 3 hours, 24 hours, and 3 days and was higher at 7 days, there were no significant differences between the 2 groups at any of the measured time points. In contrast eNOS mRNA was up- regulated in both groups during the first 3 days of reperfusion. At day 7 the eNOS mRNA was reduced to below normal in both groups (Fig. 2C). There was no significant difference between the 2 groups at any of the measured time points.

Dynamics of NOS Protein During Reperfusion

The Western blotting identified bands consistent with iNOS protein expression in both groups. Definite stainings were observed in both groups from 3 hours to 7 days of reperfusion, with a maximum at 24 hours (1249.4% 199.6% of normal in controls and 581.3% 120.3% increase in the 1400W group) (Fig. 3A). Compared with controls the iNOS protein level in the 1400W group was significantly lower at 3 (p < .05) and 24 hours (p < .01). At 14 days the iNOS protein level returned to normal in both groups. Subsequently iNOS bands were either not detectable or present only in trace amounts.

The protein levels of nNOS and eNOS were reduced slightly in both groups at 3 hours of reperfusion. The levels of nNOS remained smaller (range: 65%-91 % of normal in controls, 55%-98% in the 1400W group) and the levels of eNOS remained greater than normal (range: 108%-145% in controls, 117%-135% in the 140OW group) in both groups throughout the remainder of the experiment (Fig. 3B, C). There was no significant difference between the 2 groups at any of the measured time points.

Histologic Evaluation

Diffuse edema and degeneration of the majority of the axons were observed in both groups at day 7 of reperfusion. Although quantitative evaluation was not performed, less-severe axon degeneration was noted in the 1400W group than in controls (Fig. 4A). By day 11 the samples contained a mixture of degenerating and regenerating axonal sprouts in both groups (Fig. 4B). A higher density of regenerating axons with greater diameter and thicker myelin, however, was found in the 1400W group. At day 42 there were no appreciable differences between the 2 groups and the population of axons showed obvious recovery.

Figure 3. The effects of 1400W on the expression of the 3 NOS proteins in rat sciatic nerve after 2 hours of ischemia and up to 42 days of reperfusion. The error bars represent the SEM. (A) iNOS protein, (B) nNOS protein, (C) eNOS protein. *p < .05 and **p < .01 compared with the water group. (A-C) [white square], Water; [black square], 1400W.

Figure 4. Cross-sections of rat sciatic nerves taken from the middle of the ischemice segments (toluidine blue, 400) (A) A water- treated nerve sample at 11 days of reperfusion; (B) a 1400W-treated nerve sample at 11 days of reperfusion; (C) a water-treated nerve sample at 21 days of reperfusion; (D) a 1400W-treated nerve sample at 21 days of reperfusion.

Discussion

Our results showed that 1400W treatment effectively promoted motor functional recovery in reperfused rat sciatic nerve after 2 hours of ischemi\a. Compared with controls there was significantly earlier recovery seen as long as 28 days of reperfusion. Earlier axonal regeneration in the 1400W group also was shown by histologic examination. Molecular biological results showed that I/R affected all 3 NOS isoforms. Inducible NOS mRNA and protein were up- regulated during the first 3 days of reperfusion. Alternatively down- regulation of nNOS and up-regulation of eNOS remained to the end of 42 days of reperfusion. Administration of 1400W significantly attenuated the increase of iNOS, but had no effect on nNOS and eNOS. Thus our results support that NO/NOS is involved in I/R injury and that early inhibition of iNOS appears to be critical in the reduction or prevention of I/R injury in the PNS.

Our results indicate that inhibition of iNOS reduces I/R injury. Motor functional recovery in the 1400W group occurred approximately 1 week earlier than that in the control group, reducing the time required for axonal regeneration of rat sciatic nerve by 25%. Our results concur with those of others who reported that NOS inhibitor diminished the I/R-mediated lipid peroxidation and decreased the superoxide dismutase level, thereby protecting nerve fibers from I/ R injury.39 It also has been reported that cerebral I/R injury is exacerbated by excessive NO from iNOS36 and is reduced by green tea catechins through inhibition of iNOS.46 Our results also parallel those found in skeletal muscle I/R injury treated with 1400W30-32 and in iNOS-knockout mice.35 Thus inhibition of iNOS appears to be a potential therapeutic strategy for the treatment of peripheral nerve I/R injury.

The finding that I/R up-regulated the expression of iNOS mRNA and protein is in agreement with the results in reperfused rat PNS,14 brain,46 and rat dorsal root ganglia after limb tourniquet ischemia.47 Overproduced NO from iNOS reacts with superoxide anion to generate toxic production of peroxynitrite, which in turn affects many cellular functions,8,48-50 subsequently leading to the destruction of myelin and axons,51,52 collapse of growth cones,53 and inhibition of neurite outgrowth.54 Our functional results and histologic findings appear to support this concept. Thus excessive NO production from enhanced iNOS appears to be a critical and detrimental factor in PNS I/R injury.

This study observed the dynamics of 3 NOS isoforms in the PNS during a long period of reperfusion. Our data showed that iNOS mRNA and protein were up-regulated during the first 3 days of reperfusion (Figs. 2A, 3A). This early up-regulation affects axonal degeneration and later regeneration. Our previous study, however, failed to identify iNOS protein at 3 hours of reperfusion.14 The difference might be related to nerve sample handling; pooled samples were used in the present study but single samples were used in the previous study, in which the small size of the nerve sample may not have been able to detect the iNOS protein because of the limited sensitivity of the measurement method. Taken together our data suggest that prevention of iNOS up-regulation may be the key point in the treatment of I/R injury in the PNS.

In contrast to iNOS, nNOS mRNA and protein were down-regulated during the first 3 days of reperfusion, suggesting that different isoforms of NOS respond differently to I/R. In the literature nNOS mRNA has been shown to decrease to nearly undetectable levels in rat I/R retina.55 Hypoxia/reoxygenation down-regulates nNOS mRNA and immunoreactivity in cultured cortical cells.56 Recently we found that exogenous NO donor promotes motor functional recovery and muscle contractile force in I/R rat sciatic nerve,35 suggesting that nNOS is beneficial in PNS I/R injury. This differs from cerebral I/ R, in which nNOS is up-regulated during reperfusion57 and is thought to be detrimental.9'40 The discrepancy might reflect the importance of the cellular and tissue source in NO/NOS regulation. Considering that the regulation of nNOS is exceedingly complex58 its biological relevance in I/R injury of the PNS requires further examination.

Our results showed up-regulation of eNOS in the sciatic nerve during reperfusion (Figs. 2C, 3C). It has been reported that cerebral ischemia up-regulates eNOS protein,57,59 which is accompanied by upregulation of the vascular endothelial growth factor,60 and that inhibition of eNOS worsens I/R-mediated hippocampal dysfunction.61 Up-regulated eNOS might be critical in maintaining cerebral blood flow and in reducing platelet activation and infarct volume in cerebral ischemia.40'62 Therefore eNOS seems to be neuroprotective in neuronal I/R injury. Because eNOS contributes only a fraction of the total neuronal NOS activity63 its relative importance in PNS I/R injury should be compared carefully with that of the other 2 NOS isoforms.

It appears that induction of iNOS and/or downregulation of nNOS mediates I/R injury in the PNS. There is a simultaneous compensatory response, however, through eNOS activation to mediate vasodilation and reduce leukocyte adhesion and platelet aggregation in reperfused tissues. The sum of NO regulation is determined by the microenvironment, cellular source, concentration, and timing of NO production from each NOS isoform.9,64,65 Considering that each NOS has a distinct source, distribution, and function, the dynamic expression of individual NOS and the balance between them in I/R injury needs to be characterized precisely during the course of nerve degeneration and regeneration.

1400W is a highly selective iNOS inhibitor (5000fold over eNOS and 200-fold over nNOS).66 Subcutaneous injection of 1400W follows the published data by us30-32 and others.67,68 Although we do not know how much of the 1400W is received by the nerve in the present study, the finding that 1400W significantly reduced iNOS expression to half or less than half of that seen in controls during the first 3 days of reperfusion but had almost no effect on nNOS and eNOS (Figs. 2, 3) reflects this selectivity. 1400W may be effective only on the higher level of iNOS because there was no significant difference in iNOS expression between the 1400W group and controls after 3 days of reperfusion. In addition the finding that motor functional recovery did not differ significantly between 3 and 10 mg/ kg of 1400W and between 1 initial injection and 2 injections per day for up to 14 days suggests that the selected dose is adequate and that early administration of 1400W is critical because the first dose of 1400W was administered 10 minutes before reperfusion. Nevertheless 1400W appears potentially valuable in the prevention of I/R injury in the PNS.

It is difficult to create complete ischemia in rat sciatic nerve because of its multiple blood supplies. In addition to tourniquet ischemia several rat models have been reported. Occlusion of the femoral artery only can reduce sciatic nerve blood flow.69-71 Use of a slipknot technique to ligate temporarily the abdominal aorta, the iliac artery, the femoral artery, and all identifiable collateral vessels supplying the sciatic nerve results in severe ischemia in the nerve, but an extensive surgery is required.72,73 Although the model we used is in fact a low-load crush model our previous studies have shown that the degree of nerve damage and the speed of functional recovery at this low compression were related significantly to the duration of the applied compression.43,44 Because the nerve is interposed between 2 metal surfaces, the pressure in our model (-13 mm Hg/mm^sup 2^) is lower than that reported by others who found that venular blood flow in the rabbit vagus nerve was reduced at 20 to 30 mm Hg applied by a chamber and total ischemia occurred at 60 to 80 mm Hg.74,75 Accordingly we have applied this model to mimic the I/R condition in a series of studies although we cannot prove directly the condition of complete ischemia.

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Sang-Jin Shin, MD, PhD, Seoul, South Korea, Wen-Ning Qi, MD, Yongting Cai, M. Rizzo, MD, R. D. Goldner, MD, J. A. Nunley II, MD, Long-En Chen, MD, PhD, Durham, NC

From the Department of Orthopaedic Surgery, Ewha Women's University Mokdong Hospital, Seoul, South Korea; and the Department of Surgery, Division of Orthopaedic Surgery, Duke University Medical Center, Durham, NC.

Received for publication November 18, 2004; accepted in revised form March 8, 2005.

Supported by an Orthopedic Research and Education Foundation research grant (W.-N.Q.) and an Aircast Foundation research grant (L.-E.C).

No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

Corresponding author: Long-En Chen, MD, PhD, Orthopaedic Research Laboratory, Box 3093, Duke University Medical Center, Durham, NC 27710; e-mail: chen0006@mc.duke.edu.

Copyright 2005 by the American Society for Surgery of the Hand

0363-5023/05/30A04-0028$30.00/0

doi:10.1016/j.jhsa.2005.03.003

Copyright Churchill Livingstone Inc., Medical Publishers Jul 2005

Source: Journal of Hand Surgery, The

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