Post on 01-Jan-2016
description
Embryonic Neural Stem Cells & Potential Anesthetic-Induced Neurotoxicity
Cheng Wang, MD., PhD
Division of NeurotoxicityNational Center of Toxicological Research (NCTR)/FDA
The views provided in this presentation may not reflect those of the FDA
Representative Anesthetics
1) NMDA Antagonists: 2) GABA Agonists: Ketamine/PCP Midazolam Nitrous oxide Propofol Baclofen3) Combination (NMDA antagonists and GABA agonists): Ketamine + Midazolam Nitrous oxide + Midazolam + Isoflurane (triple anesthetic drug protocol)
4) Narcotics: Fentanyl (Control) Fentanyl is an important control because its mechanism of
action does not involve NMDA or GABA systems
INTRODUCTION
A great deal of concern has recently arisen regarding the safety of anesthesia in infants and children.
There is mounting and convincing preclinical evidence in rodents and non-human primates that anesthetics in common clinical use are neurotoxic to the developing brain.
The clinical relevance of anesthetic neurotoxicity is an urgent matter of public health.
Recent advances in our understanding of stem cell biology and neuroscience have opened up new avenues of research for detecting anesthetic-induced neurotoxicity and developing potential protection/prevention strategies against anesthetic-induced neuronal injury.
DIV 2 DIV 4 DIV 6 DIV 8
Rat Embryonic Neural Stem Cells
• Embryonic neural stem cells were harvested from cortical tissue of timed pregnancy embryonic day 14 Sprague-Dawley rats.
• plate cells at 96-well plate or petris at a concentration of one million cells per ml.
• Grow in serum-free N2 medium containing bFGF, EGF, NT3 and PDGF (change medium every 3 days).
• perform experiments on Day In Vitro (DIV) 8.
A B
Contrast phase Nestin
Rat Embryonic Neural Stem Cell Culture(Day-In-Vitro 8)
A
Nestin
Rat Embryonic Neural Stem Cell Culture(Day-In-Vitro 8)
B
Nestin + EdU
Propofol
2,6-diisopropylphenol
Propofol (marketed as Diprivan) is a hypnotic agent.
Propofol, a GABA receptor agonist, is a widely used anesthetic agent for induction and maintenance of anesthesia for adults and children.
Growing body of data suggest that exposure to anesthetics during certain periods of development has long-term deleterious effects.
At the cellular level, there is evidence that anesthetic agents induce cell death, cause synaptic remodeling, and alter morphology of the developing brain.
Objective & Specific Aims
Using embryonic neural stem cells, it is of considerable interest to determine:
1) How they respond to stress, e.g., prolonged (time-course) propofol exposure.
2) How propofol exposure (dose response) directs/signals these cells to undergo apoptosis or necrosis.
3) How their proliferation rate is affected by short- or prolonged anesthetic exposure.
4) How their fate, e.g., differentiation from progenitor cells to neurons or glial cells, is affected.
5) How the anti-oxidant agents affect anesthetic-induced neurotoxicity.
6) Clinically relevant protective strategies against anesthetic-induced developmental neural damage.
LD
H r
elea
se (
O.D
. 40
5 n
m)
0uM
10uM
50uM
100u
M
300u
M
600u
M
0.0
0.5
1.0
1.5
2.0
2.5
*****
LDH Release(Propofol; 24-hour Exposure)
MT
T O
.D.
(590
nm
)
0uM
10uM
50uM
100u
M
300u
M
600u
M
0.0
0.5
1.0
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2.0
****
*** *** ***
MTT-Assay(Propofol; 24-hour Exposure)
**a
MT
T O
.D. (5
90 n
m)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
MT
T O
.D. (5
90
nm
)
0.0
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0.2
0.3
0.4
0.5
3-hour Exposure
6-hour Exposure
Control Propofol (10 µM) propofol (50 µM) Propofol (100 µM)
A
B
Propofol Exposure (24-hour)
A B
Control Propofol (50 µM; 24 hours)
A B
Control Propofol (50 µM)
TUNEL-Assay
Scatter Plot (Sorting)Cellometer Vision
Control Propofol (50 µM; 24 hours)
EdU-DAPPI Staining
Control Propofol (50 µM; 24 hrs)
A
E
B
F
C D
EdU
DAPPI
Edu-DAPPI
Neural Stem Cell Proliferation(Propofol; 24-hour Exposure)
% o
f E
dU
Control
50uM
100u
M
0
20
40
60
80
*
8-o
xo
-dG
Co
nc
en
trati
on
(n
g/m
L)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
*
Control Pro 3 hours Pro 6 hours Pro 24 hours
Pro = Propofol (50 µM)
Assessment of Mitochondrial Membrane Potential
Control Propofol-exposed
8-o
xo
-dG
Co
ncen
trati
on
(n
g/m
L)
0.0
0.5
1.0
1.5
2.0 ***
*
8-oxo dG ELISA-Assay(Propofol; 24-hour Exposure)
Control Propofol (50µM) Propofol+L-Ca L-Ca alone
L-Ca = Acetyl-L-Carnitine (10 µM)
Ce
ll D
ea
th D
ete
cti
on
EL
ISA
(O
.D. 4
95
nm
)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
*
SUMMARY
Prolonged propofol exposure at clinically relevant concentration induces adverse effects on embryonic neural stem cells.
Propofol-induced cell damage is most probably apoptotic in nature. Data from the EdU assay suggest that 24 h propofol (50 µM) can slow or stop neural stem cell division/proliferation.
The presence of elevated levels of 8-oxo dG and its analogs in the culture medium suggest oxidative damage due to an increased generation of reactive oxygen species (ROS).
Co-administration of acetyl-l-carnitine effectively blocks at least some of the toxicity of propofol, presumably by reducing ROS generation or increasing ROS scavenging.
Anion
Cation
unbalance
ROSROS
AnionsCations
AnionsCations
AnionsCations
Propofol
GABA-R
GABA-R
caspasescaspases
cell deathcell death
cytochrome c
cytochrome c
Mitocondrion
DNA Fragmentation
Chromatin Condensation
DisturbedCell Cycle
DFF40/CAD
L-Carnitine
Ca2+
G1
S
M
G2
Excitatory-R
Neural Stem Cell
ACKNOWLEDGEMENTS
NCTR/FDA • Fang Liu
• Natalya Sadovova
• Charles Fogle
• Xuan Zhang
• Shuliang Liu
• Deborah Hansen
• Merle G. Paule
• William Slikker Jr.
• Joseph Hanig
• David Jacobson-Kram
• William Rodriguez NICHD,CDER, NCTR
CDER/FDA