olafa

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Jul 26, 2007
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Ineresting article- I tried to post the whole article but its too large. Images wouldn't cut-paste and the entire text article is too long to paste.... Anyhow, something intersting to think about as we inject those knees- it was published in Journal of Bone and Joint Surgery 2010.


Apoptosis and Mitochondrial Dysfunction in
Human Chondrocytes Following Exposure to
Lidocaine, Bupivacaine, and Ropivacaine
By Valentina Grishko, PhD, Min Xu, MD, Glenn Wilson, PhD, and Albert W. Pearsall IV, MD
[FONT=AdvOT255b5711.I][FONT=AdvOT255b5711.I]Investigation performed at the Department of Orthopaedic Surgery, University of South Alabama, Mobile, Alabama.


.Background:
Several mechanisms have been proposed to explain toxicity of local anesthetics to chondrocytes, including
the blockade of potassium channels and mitochondrial injury. The purposes of this investigation were to study the
effects of lidocaine, bupivacaine, and ropivacaine on human chondrocyte viability and mitochondrial function in vitro and
to characterize the type of cell death elicited following exposure.
Methods:


Primary chondrocyte cultures from patients with osteoarthritis undergoing knee replacement were treated
with saline solution and the following concentrations of local anesthetics: 2%, 1%, and 0.5% lidocaine, 0.5% and 0.25%
bupivacaine, and 0.5% and 0.2% ropivacaine for one hour. Cell viability and apoptosis were measured by flow cytometry at
twenty-four hours and 120 hours after treatment. Nuclear staining and caspase 3 and 9 cleavage assays (Western blot)
were used to further establish the induction of apoptosis. Mitochondrial dysfunction was evaluated by the accumulation
of mitochondrial DNA damage (quantitative Southern blot), changes in adenosine triphosphate production (bioluminescence
kit), and mitochondrial protein levels (Western blot analysis).
Results:


Exposure of primary human chondrocytes to a 2% concentration of lidocaine caused massive necrosis of
chondrocytes after twenty-four hours, 1% lidocaine and 0.5% bupivacaine caused a detectable, but not significant,
decrease in viability after twenty-four hours, while 0.5% lidocaine, 0.25% bupivacaine, and both concentrations of
ropivacaine (0.5% and 0.2%) did not affect chondrocyte viability. Flow cytometry analysis of chondrocytes 120 hours after
drug treatment revealed a significant decrease in viability (p < 0.05) with a concomitant increase in the number of
apoptotic cells at all concentrations of lidocaine, bupivacaine, and ropivacaine analyzed, except 0.2% ropivacaine.
Apoptosis was verified by observation of condensed and fragmented nuclei and a decrease in procaspase 3 and 9 levels.
Local anesthetics induced mitochondrial DNA damage and a decrease in adenosine triphosphate and mitochondrial
protein levels.
Conclusions:


Lidocaine, bupivacaine, and ropivacaine cause delayed mitochondrial dysfunction and apoptosis in cultured
human chondrocytes.
Clinical Relevance:


Local anesthetics cause deleterious effects on human chondrocytes in vitro. The results of the
present study establish a basis for the further investigation of local anesthetic toxicity in an in vivo system.
D


rugs that block voltage-gated sodium channels have
been a mainstay of pain medicine since the introduction
of cocaine more than 120 years ago. At the
same time, these local anesthetics are known to have both
beneficial and adverse effects on a variety of cellular activities,
such as wound-healing, thrombosis, and inflammatory responses,
and to cause cellular toxicity.
Intra-articular injection of local anesthetics is widely
used to control pain following arthroscopic surgery. Currently,
bupivacaine is the best studied and most commonly used agent
during this type of surgical procedure

1. However, chondrotoxicity
has been demonstrated after treatment with bupivacaine
2,3


as well as other anesthetics4-6. Moreover, there are a
number of reports concerning local anesthetic toxicity on
Disclosure:


The authors did not receive any outside funding or grants in support of their research for or preparation of this work. Neither they nor a
member of their immediate families received payments or other benefits or a commitment or agreement to provide such benefits from a commercial
entity.
609

other cell types, including the induction of necrosis and apoptosis


7-9. Previous studies have been related to the immediate or



short-term effects of bupivacaine or lidocaine. The long-term
effects of bupivacaine, lidocaine, or ropivacaine on human
chondrocytes are not known.
The exact mechanisms of local anesthetic toxicity have
not been fully elucidated. It appears that the mechanism of toxicity
is probably unrelated to the primary action of all local anesthetics,
the blockade of the voltage-gated sodium channels

10,11.
Recent investigations have shown that local anesthetics have
an influence on potassium and calcium channels

12-16.
Several studies have suggested that local anesthetics may
affect mitochondrial energetics, and mitochondrial insults can
induce either apoptosis or necrosis, with less severe injuries
leading to apoptosis, a form of programmed cell death

17-19. It is
well established that various toxic stimuli can unbalance the
electron transport chain and enhance endogenous free radical
production in mitochondria

20. Reactive oxygen species pro-
Fig. 1
Scatterplots of flow cytometry determinations of the numbers of necrotic, apoptotic, and viable cells twenty-four hours after a one-hour
exposure to the following (from left to right, from top to bottom): saline solution, 2% lidocaine, 1% lidocaine, 0.5% lidocaine, 0.5%
bupivacaine, 0.25% bupivacaine, 0.5% ropivacaine, and 0.2% ropivacaine. Propidium iodide fluorescence (PIPE-A; ordinate) was plotted
against annexin-V fluorescence (FITC-A; abscissa). Quadrant 3 (Q3) shows live cells, quadrants 1 and 2 (Q1 and Q2) show necrotic cells,
and quadrant 4 (Q4) shows apoptotic cells. Note the presence of necrotic cells and the absence of apoptotic cells at twenty-four hours

after exposure to local anesthetics.
induces damage to lipids, proteins, and nucleic acids in
mitochondria. Reactive oxygen species-induced mitochondrial
DNA (mtDNA) damage and mutations lead to the synthesis of
functionally impaired respiratory chain subunits, causing respiratory
chain dysfunction and augmented reactive oxygen
species production​
21,22. This vicious cycle has been proposed to
cause an exponential increase in mtDNA damage and mutations
over time, resulting in functional failure and cell death
23.
We hypothesized that the accumulation of mtDNA damage,
mediated by blockade of potassium channels, is a key factor
contributing to the development of chondrocyte toxicity induced
by local anesthetics.
The purposes of this study were to compare the effects of
lidocaine, bupivacaine, and ropivacaine on human chondrocyte
mitochondrial function and viability in vitro and to characterize
the type of cell death elicited following exposure to local
anesthetics.

Materials and Methods​
Cartilage Specimens and Chondrocyte Cultures​
W​
e used cartilage (from both femoral condyles and tibial
plateaus) obtained from patients with osteoarthritis
who were an average (and standard deviation) of 53
± 16 years
old and undergoing total knee replacement. The cartilage was
removed by cuts through the cancellous bone during preparation
for a total knee arthroplasty. The total time from cartilage
harvest to culture initiation varied from one to three
hours. After specimen inspection, all cartilage was removed for
culture initiation except for that from surrounding areas devoid
of cartilage, which likely contained only dead cells.
Primary chondrocyte cultures were generated by overnight
digestion of minced cartilage samples with 5 mg/mL of
collagenase B (Roche, Indianapolis, Indiana) in Dulbecco's
Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12;
Invitrogen, Carlsbad, California) supplemented with 10% fetal
bovine serum (FBS; HyClone, Logan, Utah) and antibiotics.
Cells were plated and used for experiments after reaching
confluence (seven to ten days). In order to preserve chondrocyte
phenotype, primary chondrocyte cultures were never passaged.
Confluent cultures were routinely checked for the presence of
collagen II by Western blot analysis with anticollagen-II antibody
to ensure that the chondrocytes studied had a normal
phenotype (data not shown).
The chondrocyte cultures generated for this study represent
a mixed population of less and more-damaged cartilage
cells, but this likely reflects what is present in osteoarthritic
cartilage. Thus, the results obtained from this study show the
overall effect of local anesthetics on osteoarthritic cartilage.
Likely the effect of these drugs on chondrocytes from advanced
lesions would be more severe than on less-damaged cells.
However, it is not technically possible to separate out sufficient
numbers of chondrocytes with high or low levels of damage for
independent study. Therefore, a different approach was used,
with each experiment carried out within cultures generated
from a single specimen, including all necessary controls. Because
there was no dramatic variation between experiments,
we believe we had uniform preparations of cultures, which
responded similarly to the introduced drugs.
Normally, about twenty to twenty-five confluent 100-
mm dishes were obtained from culture preparations, and after
drug exposure each dish provided a yield of about 20 to 30
mg
of DNA, which was sufficient to do three or four Southern blot
analyses. For protein isolation, smaller dishes were used, and
ATP (adenosine triphosphate) assays required only 10,000
cells. For example, with use of a single 100-mm dish, cells were
counted after trypsinization; 10,000 cells were lysed to perform
an ATP assay, about 100,000 cells were used for flow cytometric
analysis, and the rest of the cells were used for the isolation of
DNA, RNA, or proteins. As mentioned above, the amount of
DNA was sufficient to do three or four Southern blot analyses,
while the total protein amount obtained by this procedure was

Fig. 2​
Viability of human chondrocytes as determined by flow cytometry
analysis at twenty-four hours, seventy-two hours, and 120 hours
after exposure to saline solution (C), 1% and 0.5% lidocaine (L)
(top panel); 0.5% and 0.25% bupivacaine (B), and 0.5 and 0.2%
ropivacaine (R) (bottom panel). The results were obtained from a
minimum of seven independent experiments. The shaded bars
represent the mean percentage of viable cells, and the I-bars
represent the standard error of the mean. An asterisk indicates a
significant difference (p < 0.05) between local anesthetic-treated
and nontreated chondrocytes.​
611
sufficient to perform ten to twelve Western blot analyses. To
performDAPI staining (4​
9,6-diamidino-2-phenylindole, Hoechst
33342; Invitrogen, Carlsbad, California), cells were seeded in sixwell
or twelve-well plates.

Drug Preparation and Exposure​
After reaching confluence, primary human articular chondrocyte
cultures were exposed for one hour to the following
concentrations of local anesthetics: 2%, 1%, and 0.5% lidocaine
(Hospira, Lake Forest, Illinois); 0.5% and 0.25%
bupivacaine (Hospira); and 0.5% and 0.2% ropivacaine
(Astra Zeneca, Wilmington, Delaware). All drugs used were
preservative-free and contained only local anesthetics dissolved
in saline solution. Control cultures were exposed to
saline solution (Hospira) under the same conditions. After
sixty minutes, cells were immediately lysed or placed in​
Fig. 3​
Scatterplots of flow cytometry determinations of the numbers of necrotic, apoptotic, and viable cells 120 hours after a one-hour exposure to
the following (from left to right, from top to bottom): saline solution, 1% lidocaine, 0.5% lidocaine, 0.5% bupivacaine, 0.25% bupivacaine,
0.5% ropivacaine, and 0.2% ropivacaine. Propidium iodide fluorescence (PIPE-A; ordinate) was plotted against annexin-V fluorescence
(FITC-A; abscissa). Quadrant 3 (Q3) shows live cells, quadrants 1 and 2 (Q1 and Q2) show necrotic cells, and quadrant 4 (Q4) shows
apoptotic cells. Note the increase of dead cells with the prevalence of apoptosis at 120 hours after exposure to local anesthetics.​
612​

normal culture medium to allow time for recovery for
twenty-four, seventy-two, and 120 hours.
To investigate whether blockade of sodium channels was
involved in mitotoxicity and cytotoxicity of local anesthetics,
we exposed some cultures to tetrodotoxin (Sigma, St. Louis,
Missouri). Tetrodotoxin is another selective blocker of sodium
channels that is widely used to study the role of these channels
in normal physiology and disease
24-26. Tetrodotoxin was diluted
in water, and chondrocytes were treated with this agent to a
final concentration of 1 or 20
mM for one hour alone or in
combination with lidocaine. Some cultures were exposed to
local anesthetics in Earle's Balanced Salts formulation media
(EBSS; HyClone), where 60 mM of sodium chloride was replaced
with 60 mM of potassium chloride. Following exposure
or recovery, cells were collected and used for the evaluation of
mtDNA repair and damage, ATP synthesis, the induction of
apoptosis, or change in concentration of specific mitochondrial
proteins.

Flow Cytometry​
The ApoScreen Annexin V apoptosis kit (SouthernBiotech,
Birmingham, Alabama), which employs fluorescein-labeled
annexin V (Annexin V-FITC) in concert with propidium iodide
to evaluate subpopulations of cells undergoing apoptosis,
was used for the current investigation​
27. At designated time
points following recovery from local anesthetic exposure,
chondrocytes were trypsinized and collected by centrifugation.
To ensure that all cells were harvested, cell culture media from
each dish was combined with the resulting cell suspension
from the same dish following trypsinization. Cells were washed
twice in cold phosphate-buffered saline solution and labeled
with V-FITC and propidium iodide for thirty minutes according
to the manufacturer's suggestions. Samples were analyzed
by a FACSDiva flow cytometry machine (Becton
Dickinson, Franklin Lakes, New Jersey) to identify apoptotic
(V-FITC-labeled), necrotic (propidium iodide-labeled), and
viable cells.

Programmed Cell Death Evaluation​
To further evaluate the involvement of apoptosis in chondrocyte
death following lidocaine, bupivacaine, or ropivacaine
exposure, primary human chondrocyte cultures were exposed
to predetermined concentrations of these drugs for sixty minutes.
After treatment, the normal growth media was replenished,
and, twenty-four, seventy-two, or 120 hours later, the
appearance of apoptosis was evaluated by the observation of
condensed and fragmented nuclei following DAPI staining.
To analyze whether caspase activation was involved in
the initiation of apoptosis following exposure to local anesthetics,
Western blot analysis with use of antibodies against
caspase 3 and caspase 9 was employed. Anti-actin antibody was
used to ensure equal loading of protein samples.
For total cellular protein isolation, cells were lysed in cell
lysing buffer (Cell Signaling Technology, Danvers, Massachusetts)
and processed according to the manufacturer's suggestions.
Chondrocyte suspensions were briefly sonicated on ice,
were centrifuged once more at 5000 g to pellet any remaining
debris, and the supernatant protein was used for Western blot
assays. The protein concentration was determined with use
of the Bio-Rad protein dye micro-assay (Bio-Rad, Hercules,
California), according to the manufacturer's recommendation.​
Mitochondrial DNA Damage Assay​
Following sixty minutes of exposure to 2%, 1%, and 0.5%
lidocaine; 0.5% and 0.25% bupivacaine; and 0.5% and 0.2%
ropivacaine, DNA was extracted from primary chondrocyte
cultures that were lysed in buffer containing 10 mM Tris-HCl
(pH 8.0), 1 mM EDTA (pH 8.0), 0.5% sodium dodecyl sulfate,
and 300​
mg/mL proteinase K (Roche) overnight. DNA
was isolated by standard phenol-chloroform extraction,
precipitated with cold ethanol, and digested overnight with

Fig. 4​
Apoptosis in human chondrocytes at 120 hours following a single
one-hour exposure to local anesthetics. The top panel represents
the DAPI staining showing the condensation and fragmentation of
chondrocyte nuclei following the induction of apoptosis as observed
in a fluorescent microscope (​
·40); the bottom panel shows
the calculation of the percentage of apoptotic cells according to
flow cytometry experiments. The results were obtained from a
minimum of six independent experiments. The shaded bars represent
the mean percentage of apoptotic cells, and the I-bars
represent the standard error of the mean. An asterisk indicates a
significant difference (p < 0.05) between local anesthetic-treated
and nontreated chondrocytes. C
= control, L or Lido = lidocaine, R
or Rop
= ropivacaine, and B or Bup = bupivacaine.

 
Last edited:

olafa

10+ Year Member
Jul 26, 2007
661
31
Status
  1. Attending Physician
the rest of it-
normal culture medium to allow time for recovery for
twenty-four, seventy-two, and 120 hours.
To investigate whether blockade of sodium channels was
involved in mitotoxicity and cytotoxicity of local anesthetics,
we exposed some cultures to tetrodotoxin (Sigma, St. Louis,
Missouri). Tetrodotoxin is another selective blocker of sodium
channels that is widely used to study the role of these channels
in normal physiology and disease​
24-26. Tetrodotoxin was diluted
in water, and chondrocytes were treated with this agent to a
final concentration of 1 or 20
mM for one hour alone or in
combination with lidocaine. Some cultures were exposed to
local anesthetics in Earle’s Balanced Salts formulation media
(EBSS; HyClone), where 60 mM of sodium chloride was replaced
with 60 mM of potassium chloride. Following exposure
or recovery, cells were collected and used for the evaluation of
mtDNA repair and damage, ATP synthesis, the induction of
apoptosis, or change in concentration of specific mitochondrial
proteins.

Flow Cytometry​
The ApoScreen Annexin V apoptosis kit (SouthernBiotech,
Birmingham, Alabama), which employs fluorescein-labeled
annexin V (Annexin V-FITC) in concert with propidium iodide
to evaluate subpopulations of cells undergoing apoptosis,
was used for the current investigation​
27. At designated time
points following recovery from local anesthetic exposure,
chondrocytes were trypsinized and collected by centrifugation.
To ensure that all cells were harvested, cell culture media from
each dish was combined with the resulting cell suspension
from the same dish following trypsinization. Cells were washed
twice in cold phosphate-buffered saline solution and labeled
with V-FITC and propidium iodide for thirty minutes according
to the manufacturer’s suggestions. Samples were analyzed
by a FACSDiva flow cytometry machine (Becton
Dickinson, Franklin Lakes, New Jersey) to identify apoptotic
(V-FITC-labeled), necrotic (propidium iodide-labeled), and
viable cells.

Programmed Cell Death Evaluation​
To further evaluate the involvement of apoptosis in chondrocyte
death following lidocaine, bupivacaine, or ropivacaine
exposure, primary human chondrocyte cultures were exposed
to predetermined concentrations of these drugs for sixty minutes.
After treatment, the normal growth media was replenished,
and, twenty-four, seventy-two, or 120 hours later, the
appearance of apoptosis was evaluated by the observation of
condensed and fragmented nuclei following DAPI staining.
To analyze whether caspase activation was involved in
the initiation of apoptosis following exposure to local anesthetics,
Western blot analysis with use of antibodies against
caspase 3 and caspase 9 was employed. Anti-actin antibody was
used to ensure equal loading of protein samples.
For total cellular protein isolation, cells were lysed in cell
lysing buffer (Cell Signaling Technology, Danvers, Massachusetts)
and processed according to the manufacturer’s suggestions.
Chondrocyte suspensions were briefly sonicated on ice,
were centrifuged once more at 5000 g to pellet any remaining
debris, and the supernatant protein was used for Western blot
assays. The protein concentration was determined with use
of the Bio-Rad protein dye micro-assay (Bio-Rad, Hercules,
California), according to the manufacturer’s recommendation.​
Mitochondrial DNA Damage Assay​
Following sixty minutes of exposure to 2%, 1%, and 0.5%
lidocaine; 0.5% and 0.25% bupivacaine; and 0.5% and 0.2%
ropivacaine, DNA was extracted from primary chondrocyte
cultures that were lysed in buffer containing 10 mM Tris-HCl
(pH 8.0), 1 mM EDTA (pH 8.0), 0.5% sodium dodecyl sulfate,
and 300​
mg/mL proteinase K (Roche) overnight. DNA
was isolated by standard phenol-chloroform extraction,
precipitated with cold ethanol, and digested overnight with

Fig. 4​
Apoptosis in human chondrocytes at 120 hours following a single
one-hour exposure to local anesthetics. The top panel represents
the DAPI staining showing the condensation and fragmentation of
chondrocyte nuclei following the induction of apoptosis as observed
in a fluorescent microscope (​
·40); the bottom panel shows
the calculation of the percentage of apoptotic cells according to
flow cytometry experiments. The results were obtained from a
minimum of six independent experiments. The shaded bars represent
the mean percentage of apoptotic cells, and the I-bars
represent the standard error of the mean. An asterisk indicates a
significant difference (p < 0.05) between local anesthetic-treated
and nontreated chondrocytes. C
= control, L or Lido = lidocaine, R
or Rop
= ropivacaine, and B or Bup = bupivacaine.

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BamHI. Prior to loading on an alkaline agarose gel for
Southern blot analysis, each sample containing 5​
mg of total
DNA was incubated with 0.1 N of NaOH to reveal singlestrand
breaks. After gel electrophoresis under alkaline conditions,
DNA was transferred by means of vacuum transfer to a
nylon membrane. Membranes were hybridized with a polymerase
chain reaction-generated radioactive specific probe
representing a part of the cytochrome-c oxidase subunit-III
human mitochondrial gene. BamHI was selected because human
mtDNA has a single restriction site for this enzyme so
that, on digestion, it linearizes the mtDNA, and hybridization
with the human mitochondrial gene-specific probe to
cytochrome-c oxidase subunit III recognizes the restriction
band of 16,569 bp, corresponding to the whole mitochondrial
genome
28. Autoradiographs were scanned for hybridization
band intensity. DNA damage was evaluated as the number of
DNA breaks per 16.6-kb fragment. Break frequency was determined
with use of the Poisson expression (s
= –lnP0, where s
is the number of breaks per fragment and P
0 is the fraction of
fragments free of breaks).

ATP Bioluminescence Assay​
Human primary chondrocyte cultures were exposed to different
doses of local anesthetics (the same as for apoptosis and
mtDNA damage studies) for sixty minutes and then were replenished
with normal media; three hours later, the ATP levels
in cells were evaluated with use of an ATP bioluminescence
assay kit (Roche). This technique is well established and uses
the ATP dependency of the light-emitting luciferase-catalyzed
oxidation of luciferin for the measurement of extremely low
concentrations of ATP.​
Western Blot Analysis​
To analyze changes in mitochondrial proteins, Western blot
analysis was employed. For total cellular protein isolation, cells
were lysed in cell lysing buffer (Cell Signaling Technology) and
processed according to the manufacturer’s suggestions. This
analysis was used to evaluate levels of subunit III of cytochromec
oxidase. This subunit is one of thirteen proteins encoded by
mitochondria, and accumulation of mitochondrial dysfunc-​
Fig. 5​
Western blot analysis of procaspase 3 and 9 levels in human
chondrocytes at 120 hours following exposure to 0.5%
and 1% lidocaine (L0.5 and L1), 0.5% and 0.2% ropivacaine
(R0.5 and R0.2), and 0.5% and 0.25% bupivacaine (B0.5
and B0.25). Note the diminished levels of procaspase 3 and
9 after higher drug concentrations. Anti-actin antibody was
used to ensure equal loading. C​
= control.

Fig. 6​
Mitochondrial DNA damage induced by local anesthetics in human chondrocytes. The left panel is a representative autoradiograph
from Southern blot analyses of mtDNA from osteoarthritic chondrocytes incubated for one hour with various doses of
lidocaine and bupivacaine. Human chondrocytes were lysed for DNA extraction following overnight digestion with collagenase B.
The decreased intensity of the hybridization bands indicates mtDNA damage. The right panel displays quantitation of the mtDNA
damage following the use of all three anesthetics. The results were obtained from a minimum of eight independent experiments.
The shaded bars represent the mean break frequency, and the I-bars indicate the standard error of the mean. Each independent
experiment was performed with use of cultures obtained from an individual cartilage specimen. Cont​
= control.

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tion should change the levels of this protein. The antibody to
this protein was obtained from MitoSciences (Eugene, Oregon).
Anti-actin antibody was used to ensure equal loading of
total protein fractions.​
Statistical Analysis​
All of the data from similar experiments on single cultures
obtained from separate patients were averaged to give an average
value relating to each analysis performed. Statistical analyses
were performed with use of the Student t test or one or two-way
analysis of variance (GraphPad Prism; GraphPad Software, La
Jolla, California), where appropriate. A difference was considered
significant when p < 0.05. The Bonferroni post hoc test was
used to determine the source of observed differences.​
Source of Funding​
There was no external funding source to support the present​
study.
 

olafa

10+ Year Member
Jul 26, 2007
661
31
Status
  1. Attending Physician
Results​
T​
wenty-four hours after exposure of chondrocyte cultures
to local anesthetics, chondrotoxicity was observed with
2% lidocaine, with an almost complete loss of viable cells due
to massive necrosis (Figs. 1 and 2). Exposure of primary
human chondrocytes to 1% lidocaine and 0.5% bupivacaine
for one hour caused a detectable, but not significant, decrease
in viability after twenty-four hours. Lower doses of 0.5% lidocaine
and 0.25% bupivacaine, as well as both concentrations
of ropivacaine (0.5% and 0.2%), did not affect
chondrocyte viability compared with saline solution controls
(Figs. 1 and 2). The decrease in viability at all of the concentrations
of local anesthetics used was primarily due to
necrosis; there was no significant increase in the number of
apoptotic cells twenty-four hours after exposure. However,
flow cytometry analysis of chondrocytes 120 hours after drug
treatment revealed a significant decrease in viability (p < 0.05),
with a concomitant increase in predominantly apoptotic cells
at all concentrations of lidocaine, bupivacaine, and ropivacaine
analyzed, except 0.2% ropivacaine (Figs. 2 and 3).We did
not include 2% lidocaine in the 120-hour study because >90%
of the chondrocytes were dead by twenty-four hours after
treatment. These results also were confirmed by the increased
number of condensed and fragmented apoptotic nuclei observed
following DAPI staining (Fig. 4). To further establish the
induction of apoptosis and the possible involvement of mitochondrial
damage in its initiation, caspase 3 and 9 activationcleavage
assays were performed. The increase in caspase-3
cleavage showed that apoptosis occurred in chondrocytes exposed
to local anesthetics, and caspase-9 cleavage showed that
apoptosis observed in the chondrocytes, following local anesthetic
exposure, involved mitochondrial dysfunction (Fig. 5).
To evaluate whether local anesthetic exposure causes
mitochondrial dysfunction in human chondrocytes in vitro,
mitochondrial DNA damage and changes in ATP and mitochondrial
protein levels were investigated. As can be seen in
Figure 6, local anesthetics caused damage to mtDNA in human
chondrocytes following one hour of exposure. Tetrodotoxin is
known to be a sodium channel blocker of a different chemical
nature, which has been extensively used by many researchers to
study mechanisms of pain and its blockade
24-26. While lidocaine,
bupivacaine, and ropivacaine belong to the amino amide
group of anesthetics and block sodium channels by interaction
with their intracellular part, tetrodotoxin (anhydrotetrodotoxin
4-epitetrodotoxin, tetrodonic acid) binds to the
extracellular side of sodium channels. To explore whether the
chondrocyte toxicity is the result of sodium channel blockade,
we studied the toxic effects of tetrodotoxin on human chondrocytes.
We treated chondrocyte cultures with 1 and 20
mMof
tetrodotoxin for one hour, isolated DNA, and subjected it to
quantitative Southern blot analysis. A tetrodotoxin concentration
of 1
mM is commonly used and has been proven to be
sufficient to block sodium channels in different cell types
29-31.
In order to ensure channel blockade, a much higher concentration
of tetrodotoxin was also used. Also, we exposed cells to

Fig. 7​
Effect of tetrodotoxin (TTX) on human chondrocyte mtDNA integrity.
Representative autoradiographs of quantitative Southern blot analysis
on DNA from cells exposed to tetrodotoxin and 2% lidocaine (L) are
displayed. Note the absence of damage in mtDNA from cultures treated
with tetrodotoxin alone. Cont​
= control.

Fig. 8​
Effect of high potassium (KCl) on mtDNA damage in human chondrocytes
following local anesthetic exposure. Note the diminished mtDNA
damage when high potassium was added during exposure to local
anesthetics. Cont​
= control, and L = lidocaine.

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a combination of tetrodotoxin and lidocaine for the same
duration. We hypothesized that, if mtDNA damage is due to
blockade of sodium channels, then tetrodotoxin and lidocaine
should have additive effects on chondrocyte mtDNA integrity.
Figure 7 displays our findings, which demonstrate that tetrodotoxin
alone, even in high concentration, did not have an
effect on chondrocyte mtDNA integrity. Additionally, there
was no additive effect of tetrodotoxin on mtDNA damage induced
by lidocaine.
It has been widely appreciated that high potassium concentrations
in the media can cause the depolarization of cell
membranes and collapse of the ionic gradients and thereby
disable proper function of potassium channels​
32. Because local
anesthetics can efficiently block potassium and calcium channels,
we hypothesized that, in the presence of high potassium,
the channel blockade can be overcome and mtDNA damage
can be ameliorated. To test this hypothesis, chondrocytes were
incubated in a modified EBSS media, in which 60 mM of
sodium chloride was replaced with 60 mM of potassium
chloride (to avoid osmotic swelling from high salt concentration);
in EBSS with 2% or 1% lidocaine; and in modified EBSS
with 2% or 1% lidocaine. Figure 8 displays the results of
quantitative Southern blot analysis in cells treated as described
above. As can be seen in this figure, there was attenuation of
mtDNA damage following treatment with 2% or 1% lidocaine
when high potassium levels were present.
Because production of energy is the main function of
mitochondria, we evaluated whether ATP levels are affected
following local anesthetic exposure. Human primary chondrocytes
were exposed for sixty minutes to the previously used
concentrations of local anesthetics for viability and mtDNA
damage studies; were replenished with normal media; and,
three hours later, were evaluated with use of an ATP bioluminescence
assay kit (Roche) to determine the ATP levels. As
can be seen in Figure 9, lidocaine and bupivacaine at higher
concentrations significantly decreased ATP levels (p < 0.05),
while ropivacaine minimally affected ATP content.
We also investigated whether mitochondrial protein levels
were affected by local anesthetic exposure. Figure 10 displays a
Western blot analysis showing the levels of the mitochondrially
encoded subunit III of cytochrome-c oxidase twenty-four
hours following exposure to different doses of lidocaine. Antiactin
antibody was used to ensure equal loading of protein
samples. As can be seen in this figure, after exposure of human
chondrocytes to 2% lidocaine, we could not detect any
cytochrome-c oxidase subunit III, while 1% and 0.5% lidocaine
diminished the level of this protein compared with the
control.

Discussion​
T​
he novel finding of this study is that exposure of human
chondrocytes to lidocaine, bupivacaine, or ropivacaine

Fig. 9​
ATP levels (in pmol/1000 cells) in human chondrocytes following exposure to 0.5%, 1%,
and 2% lidocaine (L), 0.5% and 0.25% bupivacaine (B), and 0.5% and 0.2% ropivacaine
(R). An asterisk indicates a significant difference (p < 0.05) between local anesthetictreated
and nontreated chondrocytes. The shaded bars represent the mean, and the I-bars
indicate the standard error of the mean. Cont​
= control.

Fig. 10​
Western blot analysis of cytochrome-c oxidase subunit III (Sub III COX)
levels in human chondrocytes following exposure to saline solution and
to 2%, 1%, and 0.5% lidocaine (from left to right).​
616​
T​
HE JOURNAL OF BONE & JOINT SURGERY d JBJ S .ORG

V​
OLUME 92-A d NUMBER 3 d MARCH 2010
A
POPTOSI S AND MITOCHONDRIAL DYSFUNCTION IN

C​
HONDROCYTES AFTER EXPOSURE TO LOCAL ANESTHETICS

in vitro causes a decrease in cellular viability and an increase in
the induction of apoptosis after the drugs have been removed.
Our results suggest that a mitochondrial pathway is involved in
this induction of apoptosis. Also, we demonstrate that the
chondrotoxicity of local anesthetics is associated with mitochondrial
dysfunction resulting from damage to the mitochondrial
genome. This leads to a decrease in energy production and,
ultimately, to cell death.
Multiple studies have described the detrimental effects
of local anesthetics on chondrocyte viability. For example,
Chu et al. evaluated the effects of administering 0.5% bupivacaine
to bovine chondrocytes in vitro. They reported that
>99% of the chondrocytes were killed in all bupivacaineexposed
cultures​
2. Gomoll et al. similarly showed histopathologic
and metabolic changes with continuous infusion of
0.25% bupivacaine with and without epinephrine in rabbit
shoulders
3. Karpie and Chu also reported chondrotoxic effects
of 1% and 2% lidocaine on bovine chondrocytes
6. Although
the use of static and alginate bead cultures, bovine
chondrocytes, or rabbit shoulders prevent a direct comparison
with human disease, these studies clearly establish the
chondrotoxicity of bupivacaine and lidocaine. Recently, chondrocyte
toxicity was reported by Piper and Kim
4 after exposure
of human cartilage explants and chondrocytes in primary culture
to bupivacaine and ropivacaine. Those investigators demonstrated
that, similar to our findings, 0.5%ropivacaine was not
toxic to human chondrocytes twenty-four hours after a thirtyminute
exposure, while the same concentration of bupivacaine
significantly decreased chondrocyte viability. However, these
investigators did not evaluate at a time point longer than
twenty-four hours after exposure and could have missed the
toxic effects of bupivacaine and lidocaine that we observed.
The complete understanding of the exact mechanisms
involved in local anesthetic toxicity to cartilage cells remains to
be fully elucidated. While some data related to the toxicity of
these drugs in other cell types have been reported, little is
known about the effects of local anesthetics on chondrocytes.
As mentioned above, several investigations have suggested that
local anesthetics may affect mitochondrial energetics, and mitochondrial
insults can induce either apoptosis or necrosis
17-19.
Mitochondrial injury is likely to be important in the toxicity of
local anesthetics, but this needs to be carefully investigated
because of the multiple cross talk and feedback loops that are
involved in the different mitochondrial death pathways. Also, it
should be noted that we did not perform studies on normal
chondrocytes obtained from healthy donors. Therefore, it is
likely that osteoarthritic chondrocytes already are stressed and
may not be able to recover as well as normal cells. In support of
this notion is our recent study, which demonstrates that mitochondrial
dysfunction was present in osteoarthritic chondrocytes

33​
. It is conceivable that similar mechanisms are involved in
the cell damage and death caused by the pathogenesis of osteoarthritis
and local anesthetics, and these effects are additive.
Recent studies have shown that local anesthetics have an
effect on mitochondrial function in different cell types: bupivacaine
at a concentration of
£1.5 mM uncouples isolated heart
muscle mitochondria, while higher concentrations inhibit respiration

19​
. Other support for mitochondrial involvement in local
anesthetic toxicity has been provided by studies of the effects of
lidocaine on neuronal cell lines. It has been shown that, in ND7
cells obtained from the rat dorsal root ganglion, lidocaineinduced
apoptosis correlated with mitochondrial membrane
depolarization, cytochrome-c release from mitochondria into
the cytosol, and activation of caspases
8. Interestingly, equimolar
Tris buffer and equipotent tetrodotoxin controls were
not toxic, indicating that neither osmotic nor sodium-blockade
effects explain lidocaine neurotoxicity. Our data support this
notion.We did not observe mtDNA damage after exposure to
tetrodotoxin.
Damage to mtDNA appears to be important in mitochondrial
pathways of apoptosis. It has been shown that the
loss of mitochondrial membrane potential, which leads to
swelling of mitochondria and release of proapoptotic factors
from mitochondria, is linked to the presence of mtDNA
damage
34. Also, it has been reported that oxidative damage to
mtDNA is significantly higher in apoptotic cells than in controls

35,36​
. In addition, the differential susceptibility of glial cell
types to oxidative stress and apoptosis correlates with increased
oxidative mtDNA damage
37. Finally, a number of studies have
shown that targeting DNA repair enzymes into mitochondria
not only can enhance the repair of mtDNA lesions but also can
increase the viability of a variety of cell types and protect
against the induction of apoptosis
38,39.
We believe that a particularly novel aspect of our investigation
is that potassium modulates some of the toxic
effects of local anesthetics. However, this finding requires
further investigation. In support of this notion are the findings
that potassium channel blockers affect the proliferation
and apoptotic behavior of human osteoarthritic chondrocytes

40​
. Also, it has been shown that bupivacaine is a very
potent blocker of potassium channels, and lidocaine also has
the ability to block potassium channels, although to a lesser
extent than bupivacaine
15,41. Potassium channel openers recently
have been studied and used as important tools to explore
the role of potassium channels in cell function. Many of
them are used as potent drugs to prevent pathologic changes
in various organs
42,43. Moreover, in a recent work presented by
Sun et al., a direct connection between mitochondrial injury
and potassium channels has been demonstrated
44. They found
that the K
ATP channel opener diazoxide prevented the dramatic
early accumulation of reactive oxygen species in the mitochondria
of ischemic spinal cord neurons by amelioration of
oxidative mitochondrial DNA damage and deletions, thereby
arresting apoptosis. We believe that similar mechanisms may
be involved in chondrocytes.
We understand that our studies only correlate mtDNA
damage, mitochondrial dysfunction, and cell death. In future
studies, we plan to transfect our cells with DNA repair enzymes
and definitely establish whether enhanced mtDNA repair, which
leads to a decreased steady state of mtDNA damage, can reduce
the toxicity caused by local anesthetics and, thus, enhance the

viability of chondrocytes following exposure to these drugs.
n
 
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olafa

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Sorry for the error-
The title is supposed to read LOCAL anesthetic NOT topical -
 

PMR 4 MSK

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So this is what the lawyers will be suing us for next...
 

specepic

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This has been a hot topic of discussion at my institution and on the AAPMR listserves. as an aside-I believe there have already been lawsuits and ads from attorneys on this--but mainly related to med pumps used in ortho joint procedures.

Anyhoo-

I've read about 15 articles on this and my conclusion if that dose/ concentration have much more to do with toxicity than anything else, like type of local. A know a lot of docs who have gone to lido only but there are as many papers refuting as supporting that move and if we start getting into blocking afferents and the effect that may have on pain from a duration perspective I still use Bup a lot. I think it is funny when someone stops using Bup and switches to 2% lido.

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