• View in gallery
    Figure 1

    Viability of chondrocytes that were harvested from the metacarpophalangeal joints of 4 healthy adult horses determined with an MTT assay after 30 (A) and 60 (B) minutes of exposure to saline (0.9% NaCl) solution and culture media (control); 0.25%, 0.125%, or 0.0625% bupivacaine; 1%, 0.5%, or 0.25% lidocaine with (LP) or without the preservative methylparaben; 1%, 0.5%, or 0.25% mepivacaine; or 0.5%, 0.25%, or 0.125% ropivacaine. Cell viability is reported as a percentage of cell viability for the control (100% [dotted line]). Each bar represents mean percentage cell viability and each error bar represents SD. *Cell viability significantly (P < 0.05) differs between LA and control.

  • View in gallery
    Figure 2

    Viability of chondrocytes that were harvested from the horses in Figure 1 determined with an LDH assay after 30 (A) and 60 (B) minutes of exposure to various concentrations of various LAs. See Figure 1 for key.

  • View in gallery
    Figure 3

    Viability of FLSs that were harvested from the horses in Figure 1 determined with an MTT assay after 30 (A) and 60 (B) minutes of exposure to various concentrations of various LAs. See Figure 1 for key.

  • View in gallery
    Figure 4

    Viability of FLSs that were harvested from the horses in Figure 1 determined with an LDH assay after 30 (A) and 60 (B) minutes of exposure to various concentrations of various LAs. See Figure 1 for key.

  • View in gallery
    Figure 5

    Viability of FLSs that were harvested from the horses in Figure 1 determined with a TB assay after 30 (A) and 60 (B) minutes of exposure to various concentrations of various LAs. See Figure 1 for key.

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Evaluation of the in vitro effects of local anesthetics on equine chondrocytes and fibroblast-like synoviocytes

Ditte M. T. AdlerFrom the Department of Veterinary Clinical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, 2630 Taastrup, Denmark.

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Jeppe F. FrellesenFrom the Department of Veterinary Clinical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, 2630 Taastrup, Denmark.

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Christoffer V. KarlsenFrom the Department of Veterinary Clinical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, 2630 Taastrup, Denmark.

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Anne S. Q. DahmFrom the Department of Veterinary Clinical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, 2630 Taastrup, Denmark.

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Abstract

OBJECTIVE

To investigate the in vitro effects of clinically relevant concentrations of the local anesthetics (LAs) bupivacaine, lidocaine, lidocaine with preservative (LP), mepivacaine, and ropivacaine on equine chondrocyte and fibroblast-like synoviocyte (FLS) viability.

SAMPLE

Chondrocytes and FLSs of the metacarpophalangeal joints of 4 healthy adult horses.

PROCEDURES

Viability of chondrocytes and FLSs was determined with 3 assays: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and trypan blue (TB) exclusion (only FLS). Viability was assessed after 30- and 60-minute exposures to 0.0625%, 0.125%, and 0.25% bupivacaine; 0.25%, 0.5%, and 1% lidocaine; 0.25%, 0.5%, and 1% LP; 0.25%, 0.5%, and 1% mepivacaine; and 0.125%, 0.25%, and 0.5% ropivacaine.

RESULTS

Viability of chondrocytes was significantly decreased with exposure to 0.25% bupivacaine, 1% lidocaine, 1% LP, 1% mepivacaine, and 0.25% ropivacaine. Viability of FLSs was significantly decreased with exposure to 0.25% bupivacaine, 1% mepivacaine, 1% LP, and 0.5% ropivacaine.

CONCLUSIONS AND CLINICAL RELEVANCE

Clinically relevant concentrations of LAs had in vitro time- and concentration-dependent cytotoxicity for chondrocytes and FLSs isolated from the metacarpophalangeal joints of healthy horses. Bupivacaine was more toxic to chondrocytes than lidocaine, mepivacaine, and ropivacaine, whereas bupivacaine, LP, mepivacaine, and ropivacaine were more toxic to FLSs than preservative-free lidocaine. Several LAs may negatively affect chondrocyte and FLS viability.

Abstract

OBJECTIVE

To investigate the in vitro effects of clinically relevant concentrations of the local anesthetics (LAs) bupivacaine, lidocaine, lidocaine with preservative (LP), mepivacaine, and ropivacaine on equine chondrocyte and fibroblast-like synoviocyte (FLS) viability.

SAMPLE

Chondrocytes and FLSs of the metacarpophalangeal joints of 4 healthy adult horses.

PROCEDURES

Viability of chondrocytes and FLSs was determined with 3 assays: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), and trypan blue (TB) exclusion (only FLS). Viability was assessed after 30- and 60-minute exposures to 0.0625%, 0.125%, and 0.25% bupivacaine; 0.25%, 0.5%, and 1% lidocaine; 0.25%, 0.5%, and 1% LP; 0.25%, 0.5%, and 1% mepivacaine; and 0.125%, 0.25%, and 0.5% ropivacaine.

RESULTS

Viability of chondrocytes was significantly decreased with exposure to 0.25% bupivacaine, 1% lidocaine, 1% LP, 1% mepivacaine, and 0.25% ropivacaine. Viability of FLSs was significantly decreased with exposure to 0.25% bupivacaine, 1% mepivacaine, 1% LP, and 0.5% ropivacaine.

CONCLUSIONS AND CLINICAL RELEVANCE

Clinically relevant concentrations of LAs had in vitro time- and concentration-dependent cytotoxicity for chondrocytes and FLSs isolated from the metacarpophalangeal joints of healthy horses. Bupivacaine was more toxic to chondrocytes than lidocaine, mepivacaine, and ropivacaine, whereas bupivacaine, LP, mepivacaine, and ropivacaine were more toxic to FLSs than preservative-free lidocaine. Several LAs may negatively affect chondrocyte and FLS viability.

Introduction

Intra-articular injections of LAs are a key part of lameness investigations in horses, but intra-articular LA administration may be associated with harmful effects. In people, continuous administration of bupivacaine through an intra-articular pain pump catheter is associated with chondrolysis,1,2 and this observation led to several in vitro investigations that confirm LAs are toxic to chondrocytes.36 Other studies indicate that bupivacaine is the most toxic, compared with ropivacaine,3,4 mepivacaine,4 and lidocaine.6 Despite extensive use of LAs in horses as part of lameness examinations, potential adverse effects of LAs in the joints of horses have been minimally studied. Two in vitro studies6,7 reveal that undiluted 2% to 3% mepivacaine, 2% lidocaine, 0.75% to 1% ropivacaine, and 0.5% bupivacaine are toxic to equine chondrocytes; a third in vitro study8 reveals 0.44% mepivacaine and 0.22% bupivacaine are toxic to the chondrocytes of cartilage explants of horses. These results corroborate the findings from in vitro studies of human3,4 and bovine5 chondrocytes. Three in vivo studies911 reveal that single intra-articular injections, which are routinely administered to horses versus continuous intra-articular infusions, induce joint inflammation and affect the metabolism of articular cartilage. Chondrocyte death is associated with cartilage degradation and osteoarthritis development.4,12,13 Articular cartilage does not have tissue macrophages and, therefore, is unable to remove cellular debris,12,14 with the result that debris from necrotic or apoptotic cells, therefore, may lead to additional cartilage damage.4,12

Recent findings of a study11 of the effects of LAs on synovial fluid biomarkers of horses show that joint inflammation and collagen degradation occur after 1 intra-articular injection of 2% lidocaine or 2% mepivacaine, thereby suggesting that LAs do not only affect chondrocytes but also synoviocytes, directly or secondarily to mediators released from the chondrocytes. However, the effect of LAs on synoviocytes largely remains unknown, with the exception of only 3 published in vitro studies.8,15,16 Prolonged exposure of the synoviocytes of rabbits15 and dogs16 to bupivacaine or lidocaine results in cell injury15 and decreased cell viability,16 and prolonged exposure of the synoviocytes of horses to 0.22% bupivacaine and 0.44% mepivacaine significantly decreases synoviocyte viability.8 Synoviocytes are responsible for the synthesis of macromolecules, including hyaluronan and collagen.17 Synoviocyte injury and death may affect joint homeostasis, instigate synovial inflammation, and initiate pathways of chondrocyte injury by stimulating release of inflammatory and catabolic mediators.15,18 Consequently, synoviocyte death may reflect an indirect pathway of cartilage injury.

The objective of the study reported here was to further investigate the in vitro effect of 3 clinically relevant concentrations of 5 LAs (bupivacaine, lidocaine with and without preservative, mepivacaine, and ropivacaine) currently available in equine practice on the viability of isolated equine chondrocytes and synoviocytes.

Materials and Methods

Sample material

Cartilage and synovial membrane were collected postmortem from both metacarpophalangeal joints of 4 horses aged 5 to 12 years and without clinical signs of orthopedic disease. Horses were evaluated subjectively for lameness on a straight line on a hard surface prior to euthanasia and were excluded from the study if they were lame or had synovial effusion of either metacarpophalangeal joint. The articular surface and synovial membrane were inspected postmortem for macroscopic evidence of cartilage degradation, including erosions and wear lines, and synovial inflammation. Horses were euthanized for reasons unrelated to the present study by captive bolt followed by exsanguination according to Danish law and regulations. The Ethical and Administrative Committee at the Department of Veterinary Clinical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen approved the study protocol (protocol No. 2019-016), and horses were included after informed consent by their owners.

Cell isolation and culture conditions

Chondrocytes and FLSs were isolated as previously described.18 Articular cartilage was harvested aseptically from the distal aspect of the third metacarpal bone. Chondrocytes were isolated at 37°C by sequential digestion in high-glucose DMEM (4.5 g/L) that contained penicillin (300 U/mL), streptomycin (100 µg/mL), and gentamicin (50 µg/mL) with added 0.1% pronase for 1 hour, followed by an 18-hour incubation with added 0.15% collagenase type II. Released chondrocytes were rinsed and cultured in a monolayer in 96-well culture plates at a density of 40,000 cells/well in an incubator at a controlled humidified atmosphere (37°C; 5% CO2) in high-glucose DMEM with 10% fetal calf serum, ascorbic acid (50 µg/mL), penicillin (300 U/mL), streptomycin (100 µg/mL), and gentamicin (50 µg/mL; day 0).

Synovial membrane was harvested aseptically from the metacarpophalangeal joint cavity. Fibroblast-like synoviocytes were isolated at 37°C by digestion in high-glucose DMEM (4.5 g/L) that contained penicillin (300 U/mL), streptomycin (100 µg/mL), and gentamicin (50 µg/mL) with added 0.15% collagenase type I for 3 hours. Released FLSs were rinsed and cultured in a monolayer in T75 flasks in an incubator with a controlled humidified atmosphere (37°C; 5% CO2) in high-glucose DMEM with 10% fetal calf serum, ascorbic acid (50 µg/mL), penicillin (300 U/mL), streptomycin (100 µg/mL), and gentamicin (50 µg/mL) until 80% cell confluence when cells were then passaged 3 times by use of 0.05% Trypsin-EDTA. After the third passage, cells were seeded in 96-well culture plates at a density of 40,000 cells/well (day 0). On day 2 of the cultures of chondrocytes and FLSs, culture media were modified to exclude serum and phenol red (ie, serum- and phenol red-free [conditioned] media).

Local anesthetics

On day 4, cells were incubated in a controlled humidified atmosphere (37°C; 5% CO2) for 30 or 60 minutes with 200 µL of 1 of 3 concentrations of 1 of 5 LAs/well of conditioned media as follows: 0.25%, 0.125%, and 0.0625% bupivacainea; 1.0%, 0.5%, and 0.25% lidocaineb; 1.0%, 0.5%, and 0.25% LP (methylparaben as the preservative)a; 1.0%, 0.5%, and 0.25% mepivacainea; or 0.5%, 0.25%, and 0.125% ropivacaine.c Each LA was diluted in up to 100 µL of saline (0.9% NaCl) solution (to 2X the final concentrations) and supplemented with 100 µL of culture media to the final concentrations. Cells not incubated with an LA (control) were instead incubated with 100 µL of saline solution plus 100 µL of culture media.

Analysis of cell viability

Cell viability was assessed immediately after 30 and 60 minutes of exposure to each concentration of LA with a mitochondrial activity assay based on MTT,d a cell membrane integrity assay based on LDH,e and for FLSs only, a TB dye exclusion assay.f The latter assay could not be performed to assess chondrocyte viability because lifting the chondrocytes from the wells was not possible without harming the chondrocytes and doing so may have negatively affected their viability. All assays were performed according to manufacturers' protocols.

For the MTT assay, 50 µL of fresh serum-free, phenol red-free culture media and 50 µL of MTT reagent were added to each well containing an LA and released chondrocytes or synoviocytes and plates were then returned to the incubator. After 3 hours, 150 µL of MTT solvent was added to each well and the plate was covered with aluminum foil and placed on an orbital plate shaker for 15 minutes at room temperature (21°C). Absorbance was then read at 590 nm with a spectrophotometer.g

For the LDH assay, wells with positive control samples were exposed to 10 µL of the assay cell lysis solution. This solution was freshly prepared and kept at 4°C. After LA exposure of each well containing released chondrocytes or FLSs, 96-well plates were centrifugedh for 10 minutes at 600 × g. From each well, 10 µL was transferred to a clean 96-well plate, and 100 µL of assay solution was added to each well. Plates were incubated at room temperature (21°C) until color of the positive control sample developed. When a color change was observed, the plate was read at regular intervals at 450 nm(reference wavelength, 650 nm)g until positive control wells reached an absorbance of approximately 2.0 at which time the entire plate was read. Incubation time was recorded for each plate.

The TB dye exclusion assay was performed for FLSs. Wells containing FLSs were rinsed twice with sterile PBS, FLSs were released with 0.05% trypsin-EDTA,f trypsin-EDTA cell suspensions were transferred to 1.5-mL tubes that were centrifuged for 5 minutes at 600 × g, and resulting supernatant was carefully aspirated. Cells were resuspended in 10 µL of culture media and mixed with 10 µL of 0.4% TB stain.f Then, 10 µL of the cell-dye suspension was transferred to a hemacytometer,i and cells were counted with a light microscope.j For each sample, the total number of cells and the number of cells with absorbed blue dye (nonviable cells) were counted.

Data analysis of cytotoxicity and cell viability

Calculations and statistical analyses of cell viability associated with each LA were based on the ODs of treated and control wells for the MTT and LDH assays, whereas cell viability was based on a count of the number of nonviable cells for the TB assay. Cytotoxic effect of each LA was calculated for the specific assays according to their manufacturers' instructions as follows:

MTT assay: ([control OD – sample OD]/control OD) × 100

LDH assay: ([sample OD – control OD]/[positive control OD – control OD]) × 100

TB assay: (No. of nonviable cells/total No. of cells) × 100

Then, percentage cell viability was determined by subtracting the calculated percentage for each LA and assay from 100%. For standardization purposes, control wells were calibrated to 100% viability, and treated wells were reported as a percentage of the calibrated control wells for each assay. Cell viability > 100% indicated a higher viability for the treated cells than the control cells.

Statistical analysis

Statistical analyses were performed with commercially available software.k Normality was assessed with the Shapiro-Wilk test. The 60-minute chondrocyte data set for the LDH assay was logarithmically transformed to normalize its distribution. Cell viability was compared between control and LA-treated cells with a 1-way ANOVA for repeated measures on normally distributed and log-transformed data and then results analyzed post hoc with the Tukey test. Data for the MTT and TB assays for FLSs after 60 minutes of LA exposure were not normally distributed and, therefore, were analyzed with the Friedman test and then analyzed post hoc with the Dunn test. Values of P < 0.05 were considered significant.

Results

Chondrocytes

MTT assay

After 30 minutes of exposure to 0.0625%, 0.125%, or 0.25% bupivacaine, cell viability did not significantly differ, compared with control, but viability was significantly (P = 0.037) lower for cells exposed to 0.25% bupivacaine versus those exposed to 0.0625% bupivacaine, resulting from a simultaneous decrease and increase of cell viability, respectively (Figure 1). Cell viability was significantly (P = 0.022) decreased after 60 minutes of exposure to 1% LP, compared with control.

Figure 1
Figure 1
Figure 1

Viability of chondrocytes that were harvested from the metacarpophalangeal joints of 4 healthy adult horses determined with an MTT assay after 30 (A) and 60 (B) minutes of exposure to saline (0.9% NaCl) solution and culture media (control); 0.25%, 0.125%, or 0.0625% bupivacaine; 1%, 0.5%, or 0.25% lidocaine with (LP) or without the preservative methylparaben; 1%, 0.5%, or 0.25% mepivacaine; or 0.5%, 0.25%, or 0.125% ropivacaine. Cell viability is reported as a percentage of cell viability for the control (100% [dotted line]). Each bar represents mean percentage cell viability and each error bar represents SD. *Cell viability significantly (P < 0.05) differs between LA and control.

Citation: American Journal of Veterinary Research 82, 6; 10.2460/ajvr.82.6.478

LDH assay

Cell viability was significantly (P < 0.001) decreased after 30 minutes of exposure to 0.25% bupivacaine, compared with control, 0.125% bupivacaine, and 0.0625% bupivacaine (Figure 2). Also, cell viability was significantly decreased after 30 minutes of exposure to 0.5% ropivacaine, compared with control (P < 0.001) and 0.125% ropivacaine (P < 0.001). After exposure to 1% LP, cell viability was significantly decreased, compared with 0.5% LP (P = 0.029) and 0.25% LP (P = 0.026).

Figure 2
Figure 2
Figure 2

Viability of chondrocytes that were harvested from the horses in Figure 1 determined with an LDH assay after 30 (A) and 60 (B) minutes of exposure to various concentrations of various LAs. See Figure 1 for key.

Citation: American Journal of Veterinary Research 82, 6; 10.2460/ajvr.82.6.478

Cell viability was significantly decreased after 60 minutes of exposure to 0.25% bupivacaine, compared with control (P = 0.005) and 0.0625% bupivacaine (P = 0.001), and significantly (P = 0.012) decreased after 60 minutes of exposure to 1% mepivacaine, compared with control (Figure 2). Cell viability was also significantly decreased after 60 minutes of exposure to 1% lidocaine, compared with control (P = 0.004) and 0.25% lidocaine (P = 0.045), and after exposure to 0.5% ropivacaine, compared with control (P < 0.001) and 0.125% ropivacaine (P < 0.001). Exposure to 0.25% ropivacaine also significantly decreased cell viability, compared with control (P = 0.047) and 0.125% ropivacaine (P = 0.001). Exposure to 1% LP significantly (P < 0.001) reduced cell viability, compared with control.

Figure 3
Figure 3
Figure 3

Viability of FLSs that were harvested from the horses in Figure 1 determined with an MTT assay after 30 (A) and 60 (B) minutes of exposure to various concentrations of various LAs. See Figure 1 for key.

Citation: American Journal of Veterinary Research 82, 6; 10.2460/ajvr.82.6.478

Fibroblast-like synoviocytes

MTT assay

After 30 minutes of exposure to 0.0625%, 0.125%, or 0.25% bupivacaine, cell viability did not significantly differ, compared with control, but viability was significantly (P = 0.017) lower for cells exposed to 0.25% bupivacaine versus 0.125% bupivacaine, resulting from a simultaneous decrease and increase in cell viability, respectively (Figure 3). Cell viability was significantly (P = 0.008) decreased after 60 minutes of exposure to 0.25% bupivacaine, compared with control, and significantly decreased after exposure to 1% mepivacaine, compared with control (P = 0.015) and 0.25% mepivacaine (P = 0.042).

LDH assay

After 30 minutes of exposure to 0.0625%, 0.125%, or 0.25% bupivacaine, cell viability was not significantly different, compared with control, but viability of cells exposed to 0.25% bupivacaine was significantly (P = 0.033) lower, compared with 0.125% bupivacaine (Figure 4). Cell viability was significantly (P < 0.001) decreased after 30 minutes of exposure to 1% mepivacaine, compared with control, 0.25% mepivacaine, and 0.5% mepivacaine. After 30 minutes of exposure to 0.5% or 0.25% ropivacaine, cell viability was significantly (P < 0.001) decreased, compared with control, and after 30 minutes of exposure to 0.5% ropivacaine, cell viability was significantly (P < 0.001) decreased, compared with 0.125% ropivacaine. Cell viability was significantly (P < 0.001) decreased after 60 minutes of exposure to 0.25% bupivacaine, compared with control and 0.125% and 0.0625% bupivacaine. After 60 minutes of exposure to 1% mepivacaine or 0.5% mepivacaine, cell viability was significantly (P < 0.001) decreased, compared with control, and after 60 minutes of exposure to 1% mepivacaine, viability was also significantly (P < 0.001) decreased, compared with 0.25% and 0.5% mepivacaine. Cell viability was significantly decreased after 60 minutes of exposure to 0.5% ropivacaine, compared with control (P < 0.001) and 0.125% ropivacaine (P = 0.02).

Figure 4
Figure 4
Figure 4

Viability of FLSs that were harvested from the horses in Figure 1 determined with an LDH assay after 30 (A) and 60 (B) minutes of exposure to various concentrations of various LAs. See Figure 1 for key.

Citation: American Journal of Veterinary Research 82, 6; 10.2460/ajvr.82.6.478

Trypan blue assay

Cell viability was significantly decreased after 30 minutes of exposure to 0.25% bupivacaine, compared with control (P < 0.001), 0.125% bupivacaine (P = 0.011), and 0.0625% bupivacaine (P < 0.001), and after 30 minutes of exposure to 0.5% ropivacaine, compared with control (P = 0.033; Figure 5). Thirty minutes of exposure to 1% LP significantly (P < 0.001) decreased cell viability, compared with control, 0.5% LP, and 0.25% LP. Cell viability was significantly decreased after 60 minutes of exposure to 0.25% bupivacaine, compared with control (P = 0.025), and after 60 minutes of exposure to 0.5% ropivacaine, compared with control (P = 0.042).

Figure 5
Figure 5
Figure 5

Viability of FLSs that were harvested from the horses in Figure 1 determined with a TB assay after 30 (A) and 60 (B) minutes of exposure to various concentrations of various LAs. See Figure 1 for key.

Citation: American Journal of Veterinary Research 82, 6; 10.2460/ajvr.82.6.478

Crystallization

During the study, the wells that contained ropivacaine appeared cloudy. As the LA solutions were aspirated from the wells, crystallization was evident in all wells that contained ropivacaine; crystallization was confirmed microscopically. The amount of crystallization appeared to correlate with the concentration of ropivacaine, with higher concentrations resulting in more crystals. No further analyses of the crystals were performed, and none of the other LAs caused crystallization.

Discussion

The present study evaluated the cytotoxic effects of 5 clinically relevant LAs after a 30- and 60-minute exposure period with 3 concentrations/LA on equine chondrocytes and FLS. The present study showed that the cytotoxic effects were time and concentration dependent; these findings are similar to those for in vitro studies4,5 of other species. Also in the present study, bupivacaine was more toxic to chondrocytes than the other LAs, which was particularly evident from the results obtained at 60 minutes with the LDH assay. This finding is corroborated by findings of previous in vitro studies,4,6 including one involving equine chondrocytes.6 That study6 also reveals that 2% lidocaine is more cytotoxic than 2% mepivacaine. Breu et al4 report that 2% mepivacaine is more cytotoxic than 0.75% ropivacaine, but differences in chondrocyte viability were not detected in the present study at more clinically relevant concentrations of lidocaine (1%), mepivacaine (1%), and ropivacaine (0.5%). For FLSs, bupivacaine, mepivacaine, LP, and ropivacaine were more toxic than preservative-free lidocaine, but the results appeared to be strongly influenced by the analytical method. The toxic effects of LAs were indicated by decreased chondrocyte viability determined with MTT and LDH assays and decreased FLS viability determined with MTT, LDH, and TB assays. These assays reflect various cytotoxic effects of the LAs, and thus, use of multiple analytical methods will yield a more complete but also a less clear picture of the ongoing cellular processes. The MTT assay assesses cellular mitochondrial activity as an indicator of cell viability,19 whereas LDH and TB assays assess cell membrane integrity and permeability by analyzing the amount of LDH released from the cell cytosol20 or the ability of the TB stain to enter nonviable cells.20

The recommended volume of LAs to inject into a horse's joint is lower than or equal to the volume of synovial fluid within the joint,2123 such that chondrocytes and FLSs are likely exposed to diluted LA in vivo versus the label concentration of LA. The degree and rate of mixing of LA and synovial fluid are unknown, but horses exposed to LAs for joint blocks during lameness examination are weight bearing and, therefore, mixing will occur. Consequent to the likelihood that an LA becomes diluted in a joint, evaluated LA concentrations were adjusted to more closely mirror the in vivo LA concentrations in a joint after intra-articular injection; therefore, results presented here may be more clinically relevant.

The pharmacokinetics of LAs after their intra-articular injection in horses remain largely unknown. In dogs, the clearance of lidocaine from the synovial fluid to the serum starts 5 minutes after its intra-articular injection in the elbow joint, with lidocaine reaching a peak serum concentration after 30 minutes in 4 of 6 dogs.24 Rapid excretion of LAs after injection is further supported by the finding that peak urinary concentration of mepivacaine occurs 2 hours after its perineural injection in horses.25 Together, these findings suggest that maximum LA concentration in the synovial fluid after intra-articular injection of an LA would be achieved in < 2 hours. Therefore, cell exposure times of 30 and 60 minutes for each LA were chosen for optimal clinical relevance.

Regarding each LA, 0.25% bupivacaine significantly decreased chondrocyte and FLS viability after 30 and 60 minutes, compared with control and lower concentrations of bupivacaine. These findings are supported by in vitro studies showing the toxic effects of 0.5% bupivacaine to human,3,4 canine,26 bovine,27 and equine6 chondrocytes and of 0.22% bupivacaine to equine synoviocytes after 2 hours of exposure.8 Bupivacaine-induced toxicity for chondrocytes and FLSs demonstrated in the present study is further corroborated by in vivo findings following continuous intra-articular administration in the shoulder joints of rabbits,28 by histopathologic changes of the synovial membrane 1 to 10 days after intra-articular injection in the stifle joints of rabbits,29 and by chondrolysis after continuous intra-articular administration in the shoulder joints of people.1,2 Previous in vitro studies3,4,6,26,27 reveal the chondrotoxic effects of 0.5% bupivacaine, the standard concentration of commercially available formulations. Results of the present study of horses showed that 0.0625%, 0.125%, and 0.25% bupivacaine were also toxic to the chondrocytes and FLSs after exposure times of 30 and 60 minutes.

Mepivacaine displayed cytotoxic effects in FLS after 30- and 60-minute exposure times to concentrations of 0.25%, 0.5%, and 1.0% and in chondrocytes after a 60-minute exposure time to 1.0% mepivacaine. These findings are supported by an in vitro study4 showing that equivalent concentrations of mepivacaine significantly reduce viability of human chondrocytes. Compared with studies of bupivacaine, few studies6,7 include an investigation of the toxic effect of mepivacaine on chondrocytes and articular cartilage; Park et al6 and Silva et al7 report that 2% mepivacaine, the standard concentration of commercially available formulations, causes a decrease in equine chondrocyte viability in vitro. Also, in recent studies,8,11 even 0.44% mepivacaine reduces the viability of equine FLS and cartilage explants, when the exposure time was extended to 2 hours, and 1 intra-articular injection of 2% mepivacaine induces joint inflammation and collagen catabolism in horses.

Preservative-free 1% lidocaine resulted in chondrocyte death after 60 minutes. This finding is supported by the results of in vitro studies, in which 2% lidocaine is toxic to human chondrocytes30 and 1% lidocaine to bovine chondrocytes.5 Additionally, 0.5 to 1% lidocaine is toxic to canine synoviocytes and chondrocytes after an exposure time of 24 hours,16 but lidocaine toxicity had not been reported for clinically relevant time points prior to the present study.

The toxic effects of l% lidocaine with the preservative methylparaben on the chondrocytes and FLSs were noted after 60 and 30 minutes, respectively, of exposure. The in vitro cytotoxic effect of lidocaine combined with methylparaben had not been previously reported despite its clinical relevance. To prolong shelf life after opening a vial and to sustain pharmaceutical stability, commercially available lidocaine formulations contain methylparaben.l Various preservatives have been associated with toxic effects on chondrocytes, but methylparaben has not been shown to independently cause chondrocyte death.31 Also, methylparaben does not induce inflammation in the joints of horses in vivo.11 However, a commercially available lidocaine formulationa that contains 2% lidocaine and 0.1% methylparaben is associated with joint inflammation and collagen catabolism in vivo.11

A ropivacaine concentration of 0.5% was toxic to equine chondrocytes at 30 and 60 minutes, and 0.25% and 0.5% ropivacaine were toxic to FLSs at 30 minutes and 0.25% ropivacaine was toxic at 60 minutes. Ropivacaine is a long-acting LA that is considered to be a less toxic alternative to bupivacaine.32 The finding of decreased chondrocyte viability following ropivacaine exposure was comparable with the finding of a study4 involving human chondrocytes; however, whereas a toxic effect on equine chondrocytes was noted with 0.25% ropivacaine in the present study, a toxic effect on human chondrocytes occurs after exposure to 0.75% ropivacaine but not to 0.5% ropivacaine. Differences in concentrations at which LAs are toxic to chondrocytes and FLSs may be explained by the use of various methodologies for determining cell viability among studies.

Ropivacaine was the only LA used in the present study that resulted in crystal formation at all concentrations. Ropivacaine crystallization has been observed at physiologic pH,33,34 whereas crystallization was only observed at above physiologic pH for bupivacaine (pH, 7.7) and lidocaine (12.9). Average pH values in the synovial fluid of healthy joints of people are approximately 7.4 to 7.7 and slightly lower in arthritic joints.35,36 To mimic in vivo conditions, cell viability assays in the present study were performed at physiologic pH, which may have been the reason for the observed crystallization, but other causes (eg, LAs are known to have crystal polymorphism with different thermodynamic stabilities and mixtures with corticosteroids) for crystallization have been described.33 Concerns related to crystallization include an effect on LA bioavailability and the body. The bioavailability of the LA may be impared,34 such that in the present study, the actual concentration of ropivacaine to which the cells were exposed may have been less because the active drug may have been partly bound to the crystals and not in the solution. This may have led to an underestimation of the cytotoxic effects of ropivacaine in the present study as well as in other studies of ropivacaine. In vivo, decreased bioavailability may also result in a decreased or less predictable anesthetic effect. Crystallization may have harmful effects in vivo, such that crystallization could cause microemboli if the drug is absorbed into the bloodstream,33 and although speculative, crystals in the joints may cause microtrauma there. However, the in vivo consequences of crystals cannot be assumed on the basis of the findings of the present in vitro study and, therefore, should be investigated.

In conclusion, the results of the present study revealed decreased in vitro viability of equine chondrocytes and FLSs at clinically relevant concentrations of LAs. The cytotoxicity of various LAs was time and concentration dependent, and the results highlighted the impact that the choice of LA may have on the viability of articular chondrocytes and FLSs of horses. The most toxic LA was bupivacaine; therefore, it should only be used with care. Mepivacaine and lidocaine were similar in their effect on cell viability. Because of ropivacaine's tendency to crystallize in vitro at physiologic pH, its use is also cautioned. The time of chondrocyte and FLS exposure to various LAs is difficult to influence in vivo, but because of the dose-dependent toxic effects, use of the lowest effective dose of an LA for intra-articular anesthesia in clinical practice is recommended.

Acknowledgments

Funded in part by Boehringer Ingelheim Denmark A/S. Consumables were provided by E-vet A/S.

None of the authors have any financial or personal relationships that could inappropriately influence or bias the content of the article.

The authors acknowledge horse owners that permitted their horses for inclusion in the study.

Abbreviations

DMEM

Dulbecco modified Eagle medium

FLS

Fibroblast-like synoviocytes

LA

Local anesthetic

LDH

Lactate dehydrogenase

LP

Lidocaine with preservative

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

OD

Optical density

TB

Trypan blue

Footnotes

a.

AstraZeneca, Cambridge, England.

b.

Skanderborg Apotek, Skanderborg, Denmark.

c.

Fresenius Kabi, Bad Homburg vor der Höhe, Germany.

d.

MTT cell proliferation assay kit (ab211091), Abcam, Cambridge, England.

e.

LDH-cytotoxicity assay kit II (ab65393), Abcam, Cambridge, England.

f.

Life Technologies, Carlsbad, Calif.

g.

Power Wave X340, BioTek Instruments, Winooski, Vt.

h.

Universal 320, Hettich Zentrifugen, Tuttlingen, Germany.

i.

Hirschmann Labogeräte, Eberstadt, Germany.

j.

Leica DM IL LED, Leica, Wetzlar, Germany.

k.

Prism, version 8.30 for Windows, GraphPad Software, San Diego, Calif.

l.

Emil Almlof, AstraZeneca, Cambridge, England: Personal communication, 2014.

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Contributor Notes

Address correspondence to Dr. Adler (dima@sund.ku.dk).