Bupivacaine Induces Short-TermAlterations and Impairment in Rat Tendons

Christine Lehner,*yz PhD, Renate Gehwolf,*z PhD, Corinna Hirzinger,y MD,Daniel Stephan,§ DI, Peter Augat,§ Prof., PhD, Mark Tauber,|| MD,Herbert Resch,y Prof., MD, Hans-Christian Bauer,*z Prof., PhD,Hannelore Bauer,{ Prof., PhD, and Herbert Tempfer,*z# PhDInvestigation performed at the Institute of Tendon and Bone Regeneration,Paracelsus Medical University, Salzburg, Austria, and the Institute for Biomechanics,Trauma Center Murnau, Murnau, Germany

Background: Toxicity of the local anesthetic bupivacaine (BV) has been a matter of debate across medical fields. Numerous invitro studies demonstrate considerable toxicity of BV on various cell types.

Purpose: This study addresses the question of how tendon tissue responds to BV in vivo and in vitro.

Study Design: Controlled laboratory study.

Methods: In vitro studies on cultured rat Achilles tendon–derived cells were performed with cell viability assays and cleaved cas-pase 3 immunocytochemistry. Quantitative reverse transcription–polymerase chain reaction, Western blotting, gelatin zymogra-phy, and a biomechanical testing routine were applied on rat Achilles tendons at 1 and 4 weeks after a single unilateralperitendinous injection of 0.5% BV. The BV-mediated cell death in tendons was estimated with terminal deoxynucleotidyl trans-ferase dUTP nick end labeling (TUNEL) staining and immunohistochemical detection of cleaved caspase 3.

Results: Treatment of rat tendon–derived cells with 0.5% bupivacaine for 10 minutes had detrimental effects on cell viability,which can be reduced by N-acetyl-L-cysteine or reduction of extracellular calcium. In vivo, single peritendinous injections ofBV caused apoptosis in endotenon cells and an increase of pro–matrix metalloproteinases-9 after 6 hours. The collagen ratioshifted toward collagen type III after 6 hours and 2 days; scleraxis messenger RNA expression was reduced by 87%. Maximumtensile load was reduced by 17.6% after 1 week.

Conclusion: Bupivacaine exerts a severe, reactive oxygen species–mediated effect on tendon cell viability in vitro in a time- anddose-dependent manner, depending on extracellular calcium concentration. Culture conditions need to be taken into accountwhen in vitro data are translated into the in vivo situation. In vivo, administration of BV elicits marked but temporary functionaldamage.

Clinical Relevance: Local anesthetics cause short-term alterations in rat tendons, which, if occurring in humans to a similarextent, may be relevant regarding decreased biomechanical properties and increased vulnerability to tendon overload or injury.

Keywords: tendon; bupivacaine toxicity; local anesthetics; biomechanics; extracellular calcium

Local anesthetics are frequently administered for perioper-ative pain management via single injections or pain pumpdelivery up to 48 hours to the site of injury.4 Local anes-thetics are routinely used also for diagnosis and treatmentof a variety of tendinopathies. Cytotoxicity of amino amidetype local anesthetics like bupivacaine (BV) or lidocainehas been reported from various cell types like neurons,myocytes, and chondrocytes.6,22,25,26 In vivo BV toxicity,however, appears to be less dramatic in muscle and dermispossibly because of the high regenerative and reparativecapacity of these tissues.11,23

It recently has been shown that BV reduces the prolifer-ative capacity and cell viability of tendon fibroblasts andtendon stem/progenitor cells in vitro.13,29

In the light of well-documented toxicity of local anes-thetics in vitro and the limited regeneration potential oftendons, bupivacaine toxicity may contribute to impairedtendon healing after surgical reconstruction as well as ten-don rupture after pain therapy. We therefore hypothesizethat BV is toxic to tendon cells in vitro and in vivo.

To this end, we studied in vitro effects of BV on primaryrat tendon cells regarding potential toxicity and potentialunderlying mechanisms. Extracellular calcium has beenshown to influence BV toxicity in nerve and muscle tis-sue.15,31 In Schwann cells, BV has been found to inducereactive oxygen species (ROS)–mediated cell death.24

Therefore, the role of extracellular calcium and a potentialinvolvement of ROS in BV toxicity on tendon cells were

The American Journal of Sports Medicine, Vol. XX, No. XDOI: 10.1177/0363546513485406� 2013 The Author(s)

1

studied in this work. To test for the in vivo effects of BV,rat Achilles tendons were analyzed for toxic effects andfor molecular alterations caused by a single BV injection.A biomechanical testing routine was used to examinepotential impairment of mechanical properties causedby BV.

MATERIALS AND METHODS

Cell Culture

Tendon-derived cells (TDCs) were isolated according toa protocol described previously by Tempfer et al.33 Achillestendons of 4 female Lewis rats (aged 4 months) were cutinto small pieces and digested overnight in Dulbecco’s mod-ified eagle medium (DMEM) (Sigma-Aldrich, St Louis,Missouri) containing 10% fetal bovine serum (FBS) and30 mg/mL collagenase type II (Gibco, Invitrogen, Lofer,Austria). The released cells were pelleted, seeded in cellculture dishes, and grown to subconfluency. Passages 1and 2 were used.

Exposure of Rat Tendon–Derived Cells to BV In Vitro

For all experiments, BV dissolved in 0.9% NaCl (pH 6.9)from Actavis (Hafnarfjordur, Iceland) was used. In clinicalpractice, BV is commonly used at a concentration of 0.5%.For all described experiments, this was the maximum con-centration used.

TDCs were incubated in BV at final concentrations of0.5%, 0.250%, 0.1%, and 0.05%. Cells were cultivated for10 and 30 minutes to show time dependence of BV toxicity.

To assess the influence of extracellular calcium on BVtoxicity, additional BV incubation experiments were per-formed with calcium-free DMEM (Sigma-Aldrich, StLouis), mixed with normal DMEM (1.8 mM) to obtain finalextracellular Ca21 concentrations of 1.8 mM, 0.9 mM, and0 mM, respectively. TDCs were incubated for 10 minutes ineach medium condition.

To test for the involvement of ROS, TDCs were incu-bated 0.5% BV (diluted with NaCl) with 2 mM of theROS-scavenger N-acetyl-L-cysteine (Sigma-Aldrich, StLouis) for 10 minutes. Subsequently, viability was moni-tored with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) and calcein acetoxymethyl ester(calcein AM) assays.

To assess apoptotic effects of BV on rat tendon cells invitro, we treated TDCs with 0.05% BV for 4 hours and

performed an immunocytochemical staining with an anti-body directed against cleaved caspase 3.

Dilution of BV

For the in vitro experiments, BV was diluted in NaCl(0.9%) at 0.5%, the solvent and concentration used in clin-ical practice, and lower. BV is insoluble in cell culturemedium at concentrations higher than 0.1%.3 For detectionof apoptosis, which is induced within 3 to 6 hours, cellswere incubated with BV diluted in DMEM at 0.05%, toavoid starvation-induced cell damage due to NaCl (0.9%)incubation.

MTT Assay

Cell viability measurements by MTT (Sigma-Aldrich,Vienna, Austria) assay were performed according to a pro-tocol described by Mosmann.21 Briefly, cells were seeded in96-well plates and grown to subconfluency. After cells weretreated with BV for 10 minutes and subsequent incubationin DMEM for 50 minutes, MTT was added to the mediumat a concentration of 0.5 mg/mL for 3 hours at 37�C. Theformazan crystals were then lysed in DMSO (Sigma-Aldrich, St Louis). Absorbance was measured with a spec-trophotometer at a wavelength of 656 nm. The results areexpressed as percentage of the untreated control.

Calcein AM Assay

After exposure of TDCs to increasing concentrations of BV,2 mM calcein AM (Sigma-Aldrich, St Louis) was added. Thenonfluorescent, cell-permeable calcein converts to thebright green fluorescent calcein when hydrolyzed by intra-cellular esterases in living cells. Fluorescence was moni-tored at an excitation/emission (Ex/Em) of 494/520 nm ina spectrophotometer.

All viability and rescue experiments were performed intriplicate and repeated 6 times.

Animal Model

All experiments were conducted according to relevantnational and international guidelines. The animal experi-ments were approved by the local ethics committee. Forthe biomechanical testing, 18 Lewis rats (9 males, 9females) received a single unilateral injection of 100 mLof 0.5% BV around the Achilles tendon of the right leg.

#Address correspondence to Herbert Tempfer, PhD, Paracelsus Medical University (PMU), Institute of Tendon and Bone Regeneration, Strubergasse21, 5020 Salzburg, Austria (e-mail: Herbert.Tempfer@pmu.ac.at).

*Paracelsus Medical University, Spinal Cord Injury and Tissue Regeneration Center Salzburg, Institute of Tendon and Bone Regeneration, Salzburg,Austria.

yDepartment for Traumatology and Sports Injuries, Paracelsus Medical University, Salzburg, Austria.zAustrian Cluster for Tissue Regeneration.§Institute for Biomechanics, Trauma Center Murnau, Murnau am Staffelsee, Germany.||ATOS Clinic, Munich, Germany.{University of Salzburg, Department of Organismic Biology, Salzburg, Austria.

One or more of the authors has declared the following potential conflict of interest or source of funding: This work was supported by the Hermann andMarianne Straniak Foundation (Switzerland), by grants R-10/02/012HIR and 05/02/008 from Paracelsus Medical University (Austria), and by the LorenzBoehler Foundation (Austria).

2 Lehner et al The American Journal of Sports Medicine

The left leg was injected with 100 mL of 0.9% NaCl. After 1and 4 weeks, Achilles tendons were subjected to a biome-chanical testing routine. Control specimens (Achilles ten-dons of the left leg) were tested in the same way.

For histological and molecular analyses, 9 female Lewisrats were peritendinously injected with 100 mL of 0.5% BVaround the Achilles tendon and sacrificed at 1 of 3 timeintervals (6 hours, 2 days, and 1 week). These time pointswere chosen to catch early events such as induction of apo-ptosis or alterations in matrix metalloproteinase (MMP)production. The contralateral leg, injected with 100 mL of0.9% NaCl, served as a control. Tendons were analyzedfor expression of cleaved caspase 3, MMP2, and MMP9by immunohistochemistry and Western blotting. In addi-tion, MMP2 and MMP9 activity was detected with gelatinzymography. Messenger RNA (mRNA) levels of collagentypes I and III and gene expression of scleraxis, a markerindicative of tendon precursor cells, were quantified byquantitative reverse transcription–polymerase chain reac-tion (qRT-PCR). All animals were allowed to continue nor-mal activity between injection and analysis.

Immunohistochemistry and TUNEL Assay

Rat Achilles tendons were fixed in 4% paraformaldehyde at4�C overnight. Tissues were processed for paraffin embed-ding, cut in 6-mm-thick sections on a microtome, and stainedwith an antibody specific for cleaved caspase 3 (Asp175) (No.9661, Cell Signaling, Hitchin, UK). Incubation with primaryantibody was performed at 4�C overnight. After treatmentwith Power Vision poly HRP-anti-rabbit IgG (ImmunoLogic,Duiven, Netherlands), the sections were incubated withDAB (3,3#-diaminobenzidine [DAB] tetra hydrochloride,Sigma-Aldrich, Vienna), counterstained with Novocastrahematoxylin dye (Leica Microsystems, Vienna, Austria),and mounted in Eukitt (Sigma-Aldrich, St Louis).

Terminal deoxynucleotidyl transferase dUTP nick endlabeling (TUNEL) staining with the in situ cell deathdetection kit, POD, from Roche (Vienna, Austria) was car-ried out according to the manufacturer’s instructions.DNAse digested sections served as positive control.

Western Blot

Lysates from rat tendons and cultured TDCs were preparedas described previously.34 Protein contents were quantifiedwith a BCA protein assay kit (Pierce, Rockford, Illinois).Equal amounts of protein were loaded per lane and sub-jected to sodium dodecyl sulfate polyacrylamide gel electro-phoresis (SDS-PAGE) with either a 7.5% or a 12.5% gel.After blotting, the polyvinylidene fluoride (PVDF) mem-brane was incubated in 5% nonfat dried milk. Immunode-tection was performed with primary antibodiesrecognizing the cleaved form of caspase 3, MMP2 (No.ab37150, Abcam, Cambridge, UK) and MMP9 (No.CLRM105, Cedarlane, Hornby, Canada) and a secondaryhorseradish peroxidase (HRP)–labeled goat anti-rabbit anti-body (Sigma-Aldrich, Vienna). Bands were visualized by useof the SuperSignal West Pico Chemiluminescent Substratefrom Pierce (Thermo Scientific, Vienna, Austria).

Gelatin Zymography

For gelatin zymography, rat Achilles tendons injected a sin-gle time with 0.5% BV were analyzed at 6 hours and 7 days.

Briefly, rat tendons were lysed in a buffer containing 20mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100,and 0.1% SDS. Equal amounts of protein were loaded on7.5% polyacrylamide gels containing 1 mg/mL gelatin.Following electrophoresis, the gels were immersed indouble-distilled water containing 2.5% Triton X-100 for30 minutes at room temperature and then incubatedwith a substrate buffer (50 mM Tris-HCl, pH 8.0, contain-ing 5 mM CaCl2). Finally, the gels were stained withCoomassie blue for 30 minutes followed by several washingsteps. Areas of proteolytic activity appeared as clear bandsagainst a dark blue background.

RNA Isolation and Quantitative RT-PCR

For isolation of RNA from rat Achilles tendons, sampleswere homogenized in PureZol Reagent (Biorad, Vienna,Austria) by use of an Ultra Thurax tissue homogenizer(Qiagen, Hilden, Germany) according to the manufac-turers’ instructions.

After photometrical quantification, 1 mg of total RNAwas transcribed into a single stranded complementaryDNA (cDNA) by use of a High Capacity RNA-to-cDNAMaster Mix (Applied Biosystems, Vienna, Austria). Quan-titative RT-PCR was done with TaqMan Gene ExpressionAssays for scleraxis, a tendon precursor marker, collagenstype I and III, and TaqMan Gene Expression Master Mix(Applied Biosystems) according to the manufacturer’sinstructions in an iCycler thermo cycler (BioRad, Vienna,Austria). DeltadeltaCT (cycle threshold) calculation wasused for quantification of relative gene expression.

Gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthine-guanine phosphoribosyltransferase (HPRT1).

Biomechanical Testing

The calcaneal bone was fixed in a custom-made device thatwas filled with Technovit (Heraeus Kulzer, Wehrheim,Germany). To correctly position the calcaneal bone, two1-mm K-wires were inserted, 1 in the frontal plane of thecalcaneal bone and 1 in sagittal plane. The tendon attach-ment area remained unembedded. Tendon fibers weresecured in a screw grip by use of sandpaper (grain size100) and superglue. Specimens were tested on a universalmaterial testing machine (Zwick, Ulm, Einsingen/Germany) at 15� loading angle at 0.1 mm/min until failureafter a preload of 0.5 N.

Force (N) was measured with a load cell of 200 N (accu-racy class 1, Gassmann und Theiss) and recorded by corre-sponding software (testXpert 1, Zwick). Failure occurredeither by fracture of the calcaneus due to the predeter-mined breaking points produced by the K-wire holes (3 atthe BV-injected side, 2 at the vehicle-injected side) or byintratendinous rupture. Only intratendinously rupturedtendons (n = 13) were included in the study.

Vol. XX, No. X, XXXX Effects of Bupivacaine on Rat Tendons 3

Statistical Analysis

All data are given as means 6 SD. Time- and dose-dependence of BV toxicity by MTT and calcein AM assay,

rescue experiments with N-acetyl-L-cysteine, experimentsassessing the influence of Ca21 on cell viability, and the bio-mechanical tests were analyzed by 1-way analysis of vari-ance (ANOVA). Results with a significant ANOVA were

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Figure 1. Calcein AM assays on rat tendon cells revealed time- and dose-dependent toxicity of bupivacaine (BV). Viability of rattendon cells (passage 1) was significantly reduced after incubation with 0.5% BV for 10 minutes followed by30 minutes of incubation with calcein AM substrate (A). Lower doses of BV caused significantly reduced cell viability after 30minutes of incubation followed by 30 minutes of incubation with calcein AM substrate (B). Values are normalized to the controlcells (A: 1 = 34,448 counts; B: 1 = 11,488 counts) *P \ .05; **P \ .01.

Figure 2. Viability/proliferation of cultured tendon-derived cells (TDCs) from rat Achilles tendon treated with bupivacaine (BV). (A)Control rat Achilles TDCs. (B) Rat Achilles TDCs treated with 0.5% BV for 10 minutes. (C) Rat Achilles TDCs incubated with 2 mMN-acetyl-L-cysteine (N-Ac) and 0.5% BV for 10 minutes. Scale bar: 50 mm. (D) Proliferation/viability of rat Achilles TDCs treatedwith 0.5% BV for 10 minutes, followed by incubation with MTT substrate in Dulbecco’s modified eagle medium (DMEM) for3 hours. For the rescue experiments, cells were coincubated with 0.5% BV and 2 mM N-Ac followed by incubation in DMEM con-taining 2 mM N-acetyl-L-cysteine. (E) Rat Achilles TDCs treated with 0.5% BV and 0.5% BV 1 2 mM N-Ac for 10 minutes andincubated with either calcein AM/phosphate buffered saline (PBS) alone or calcein AM/PBS containing 2 mM N-acetyl-L-cysteinefor 30 minutes. (F) Viability/proliferation of rat TDCs treated with 0.1% BV in media containing decreasing amounts of calcium for10 minutes as evidenced by an MTT assay. *P \ .05; **P \ .01; C, control.

4 Lehner et al The American Journal of Sports Medicine

corrected for multiple comparisons by use of the Tukey HSDtest. The qRT-PCR results were analyzed by Student t test.P values �.05 are considered significant.

RESULTS

In vitro, BV was found to be toxic to rat TDCs in a concentra-tion- and time-dependent manner (Figure 1). Treatment ofcells with 0.5% BV in NaCl for 10 minutes reduced cell via-bility significantly by 39%. After 30 minutes, viability wasalmost halved (49%) by already half the concentration(0.25% BV) and was reduced by 75% at 0.5% BV, as evi-denced by calcein AM assay (Figure 1B). Both addition ofthe ROS scavenger N-acetyl-L-cysteine and reduction ofextracellular calcium attenuated the toxic effect of BV ontendon cells significantly (Figure 2).

Exposure of rat TDCs to 0.05% BV for 4 hours resulted ina notable increase (11.7% 6 3.4%) of apoptotic cells as evi-denced by cleaved caspase 3 immunoreactivity (Figure 3).

In vivo, a single peritendinous injection of 0.5% BVaround the Achilles tendon of rats led to an increase in pro-MMP9 levels after 6 hours, as demonstrated by gelatinzymography and Western blotting (Figure 4, A and B), whichdeclined to almost control levels within 1 week (Figure 4A).

Enzyme activity and protein levels of MMP2 remainedunchanged after BV injection (Figure 4, A and C).

Concomitantly, a marked increase in cleaved caspase 3expression within the first 6 hours of treatment could bedetected, which returned to almost control levels after 2days (Figure 4D).

In BV-injected tendons, an elevated number of caspase3–positive cells were present compared with control ten-dons (5.26% 6 2.20% after 6 hours; 0.25% 6 0.59% in con-trols, 0.57% 6 1.04% after 1 week). The highest amounts ofcaspase 3–positive cells were detectable after 6 hours butdeclined within 1 week (Figure 5, A-C). More than 99% ofcells immunoreactive for cleaved caspase 3 were located

at the border area and within the connective tissue of theinjected tendons (arrows).

This distribution pattern could be confirmed by TUNELassay, with 5.2% 6 3.1% apoptotic cells after 6 hours and0.18% 6 0.74% after 1 week (Figure 5, D-F).

The qRT-PCR analysis showed that a shift in the colla-gen III/collagen I ratio occurred after BV treatment, withthe highest collagen III mRNA levels detectable 2 daysafter injection of BV (control, 10.58 6 1.04; 6 hoursafter BV, 54.11 6 7.93; 2 days after BV, 152.31 6 7.46)(Figure 6A).

Six hours after BV injection, scleraxis mRNA level wasdecreased by 89%. This reduction persisted throughout2 days after injection (control, 0.6834 6 0.0335; 6 hoursafter BV, 0.072 6 0.007; 2 days after BV, 0.1594 6

0.0078) (Figure 6B).One week after BV injection, load to failure was reduced

by about 17.6% but was restored after 4 weeks (Figure 6C).Five of 18 tendons failed by fracture of the calcaneus. Only

Figure 3. Rat tendon–derived cells treated with 0.05% bupi-vacaine (in Dulbecco’s modified eagle medium) for 4 hoursare immunoreactive to an antibody against cleaved caspase3 (red). Nuclei are stained with 4#,6-diamidino-2-phenylindole(DAPI) (blue). Stained cells have fragmented nuclei (detail,DAPI channel only, white arrow) (A). In the untreated cells,\0.1% of all cells are immunoreactive (B). Scale bar:200 mm.

Figure 4. A single injection with 0.5% bupivacaine led to anincrease in pro–matrix metalloproteinase (MMP)-9 levels(white arrow) after 6 hours as evidenced by gelatin zymogra-phy (A). Western blot analysis shows that pro-MMP9 expres-sion was increased 6 hours and 2 days after injection (B),whereas MMP2 remained unaltered (C). Cleaved caspase 3was detectable by Western blot 6 hours after injection butreturned to almost control levels after 1 week (D). b-actinserved as loading control (E).

Vol. XX, No. X, XXXX Effects of Bupivacaine on Rat Tendons 5

intratendinously ruptured tendons (n = 13) were includedin the study.

DISCUSSION

The discussion about potential toxicity of local anestheticshas been fuelled by several recent publications describing

BV-induced cell death in various cell types invitro.6,13,14,16,17,26,36,38

In this study, we show that BV severely affects TDCs invitro, whereas administration of BV in vivo causesa marked albeit transient effect.

Our results are in line with earlier findings demonstrat-ing a concentration-dependent cytotoxic effect of BV on cel-lular components of the musculoskeletal system. Human

Figure 5. Histological sections of rat Achilles tendons stained for apoptotic cells by use of (A-C) cleaved caspase 3 immunohis-tochemistry and (D-F) TUNEL assay. (A and D) Control animals were injected with 0.9% NaCl. (B and E) Sections of tendons fixed6 hours after a single peritendinous injection with 100 mL of bupivacaine (BV; 0.5%). Black arrows in (B) indicate cells positive forthe expression of cleaved caspase 3; inset in (E) shows a magnification of labeled cells. (C and F) Sections of tendons fixed 1week after BV injection.

Figure 6. Quantitative reverse transcription–polymerase chain reaction analysis of rat Achilles tendons treated with 100 mL of BV (peri-tendinous injection) shows that collagen III expression was increased, and the ratio of collagens type I and III was altered 6 hours and2 days after injection (A). Also the expression of scleraxis messenger (mRNA) was reduced (B). Scleraxis gene expression was nor-malized to the expression of hypoxanthine-guanine phosphoribosyltransferase (HPRT1) and glyceraldehyde-3-phosphate dehydroge-nase (GAPDH) (line). Load at failure (normalized to contralateral failure load) of the Achilles tendon was significantly (P \ .05, n = 13)reduced 1 week after single peritendinous BV injection but was completely recovered at week 4 (C).

6 Lehner et al The American Journal of Sports Medicine

and bovine chondrocytes were shown to be severely dam-aged by BV in a concentration- and time-dependent man-ner in vitro and in vivo.6,7,18 Similarly, treatment with0.5% BV reduced cell proliferation of human tendon fibro-blasts and decreased cell viability of human hamstring ten-don stem/progenitor cells in vitro.13,29

A correlation between BV toxicity and extracellular cal-cium was described by Hung and colleagues,15 showingthat increasing amounts of calcium chloride prolong theanalgesic effect of BV but also lead to histopathologicchanges in rat sciatic nerve. Removal of extracellular cal-cium attenuated morphological changes in BV-treated iso-lated rat soleus muscles within the first 2 hours oftreatment.31

Exposure of articular cartilage to calcium-free media invitro has a significant chondroprotective effect within thefirst hours of mechanical injury.1 Considering that thisprotective effect is observed only during the first hours oftreatment, it may be concluded that the decrease of cal-cium levels does not impede ultimate cell death but elicitsa shift from immediate necrosis to delayed apoptosis.

Our data provide evidence that the cytotoxic effect is atleast partly mediated by ROS signaling. Addition of theROS scavenger N-acetyl-L-cysteine rescues BV-treatedTDCs to a notable degree, thereby preventing immediatenecrosis. BV has been described to induce ROS-mediatedapoptosis in Schwann cells and the human neuroblastomacell line SH-SY5Y.19,24 Moreover, Cela et al5 demonstratedthat BV uncouples the mitochondrial oxidative phosphory-lation and enhances ROS production in human hepatomacells (HepG2) and murine skeletal myoblasts (L6) in vitro.In primary human skeletal muscle cells, N-acetylcysteinewas shown to protect against BV-induced sarcoplasmic/endoplasmic reticulum stress and apoptosis.10 A negativecorrelation between ROS production and viability in BV-exposed fibroblasts was reported by Fedder and colleagues.8

MMP2 and MMP9 are the most important collagen typeI–degrading MMPs. We show that pro-MMP9 levelsincrease 6 hours after BV injection but decline to normallevels within 1 week (Figure 4). Because MMP9 expressionis known to be induced by ROS, we hypothesize that thiselevated expression of pro-MMP9 is the result of a BV-induced increase of ROS in rat tendon tissue.20,39

The finding that BV induces apoptosis both in tendon tis-sue and in cultivated TDCs agrees well with other reportsshowing that BV causes caspase 3 activation in variouscell types including neuronal and renal cells.17,24,25,35

Regarding the extracellular matrix, we observed a shiftin the collagen III/I mRNA ratio as early as 6 hours afterBV treatment, with the proportion of type 3 collagenexpression increasing over the following 2 days. Becausecollagen III–rich fibers are known to be smaller in diame-ter (\60 nm) and less resistant to mechanical load,28,37

the shift toward collagen III may partly explain thereduced tensile function observed after 1 week. Reducedexpression of scleraxis mRNA shows that predominatelytendon precursor cells are affected by BV.

Although a number of in vitro studies have demon-strated the toxicity of BV treatment, detrimental effectsof BV in vivo appear to be much less pronounced.

High concentrations of BV (.4%) were observed tocause local cytotoxicity including histopathologic altera-tions in vivo.32

A similar effect is caused by BV administration over a longtime period (48 hours).4,12 Despite a plenitude of in vitrostudies demonstrating the myonecrotic effect of BV, onlya few cases of myotoxic complications have been observedin patients after local anesthetic administration, particularlyin the context of repeated drug application.22,40 The highregenerative activity of skeletal muscle fibers after BV treat-ment described by many authors may account for this.2,27

Here we show that BV predominately affects cells in theloose endotenon tissue separating adjacent tendon fas-cicles, as demonstrated by detection of cleaved caspase 3(Figure 5).

Biomechanical testing suggests that impairment of ten-sile function is transient. Recently, Friel et al9 reported noimpairing effects of continuous subacromial bupivacaineinfusion on rabbit rotator cuff tendons after 2 and 4 weeks.We observed alterations in extracellular matrix (ECM)composition and reduced tensile function 2 days and 1week, respectively, after BV injection, which was restoredafter 4 weeks. Therefore, we conclude that BV elicits onlya short-term impairing effect on tendons in this rat model.

Limitations of the study are that experiments were per-formed in the rat model in order to match biomechanicaldata with molecular and histological findings. Transferringdata from the rat model to humans requires caution. Readersshould consider differences in BV concentrations effectivelyreaching the site of action due to differences in clearanceand diffusion rates, tendon dimensions, and tendon anatomy.

CONCLUSION

BV exerts a severe, ROS-mediated effect on tendon cell via-bility in vitro in a time- and dose-dependent manner, poten-tiated with a greater level of extracellular calcium. Cultureconditions therefore need to be taken into account when oneis translating in vitro data into the in vivo situation. In vivo,BV has impairing effects on cells within areas of loose con-nective tissue and elicits considerable although temporaryfunctional damage. With this animal study we show thatlocal anesthetics cause short-term alterations in tendons,which, if they occur in humans to a similar extent, may berelevant regarding decreased biomechanical propertiesand increased vulnerability to tendon overload or injury.

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8 Lehner et al The American Journal of Sports Medicine