Embryonic Neural Stem Cells & Potential Anesthetic-Induced Neurotoxicity Cheng Wang, MD., PhD...

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

1.5

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

0.1

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