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
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.
[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.
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).
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.
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
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.
entity.
609
other cell types, including the induction of necrosis and apoptosis
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
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
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
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
electron transport chain and enhance endogenous free radical
production in mitochondria
20. Reactive oxygen species pro-
after exposure to local anesthetics.
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 disease24-26. 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.
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
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
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 death23.
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.
cause an exponential increase in mtDNA damage and mutations
over time, resulting in functional failure and cell death23.
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
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.
611analysis 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.
sufficient to perform ten to twelve Western blot analyses. To
performDAPI staining (4
performDAPI staining (4
9,6-diamidino-2-phenylindole, Hoechst
33342; Invitrogen, Carlsbad, California), cells were seeded in sixwell
or twelve-well plates.
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
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.
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 disease24-26. 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
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.
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.
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
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
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 (
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.
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:
