Embryonic Neural Stem Cells & Potential
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Embryonic Neural Stem Cells & Potential Anesthetic-Induced Neurotoxicity. Cheng Wang, MD., PhD Division of Neurotoxicity National Center of Toxicological Research (NCTR)/FDA. The views provided in this presentation may not reflect those of the FDA. Representative Anesthetics.

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Embryonic neural stem cells potential anesthetic induced neurotoxicity

Embryonic Neural Stem Cells & Potential

Anesthetic-Induced Neurotoxicity

Cheng Wang, MD., PhD

Division of Neurotoxicity

National Center of Toxicological Research (NCTR)/FDA

The views provided in this presentation may not reflect those of the FDA


Embryonic neural stem cells potential anesthetic induced neurotoxicity

Representative Anesthetics

1) NMDA Antagonists: 2) GABA Agonists:

Ketamine/PCP Midazolam

Nitrous oxide Propofol

Baclofen

3) 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


Embryonic neural stem cells potential anesthetic induced neurotoxicity

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.


Embryonic neural stem cells potential anesthetic induced neurotoxicity

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.

DIV 2 DIV 4 DIV 6 DIV 8


Embryonic neural stem cells potential anesthetic induced neurotoxicity

Rat Embryonic Neural Stem Cell Culture

(Day-In-Vitro 8)

B

A

Contrast phase

Nestin


Embryonic neural stem cells potential anesthetic induced neurotoxicity

Rat Embryonic Neural Stem Cell Culture

(Day-In-Vitro 8)

B

A

Nestin

Nestin + EdU


Embryonic neural stem cells potential anesthetic induced neurotoxicity

Propofol

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.

2,6-diisopropylphenol

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.


Embryonic neural stem cells potential anesthetic induced neurotoxicity

Objective & Specific Aims

  • Using embryonic neural stem cells, it is of considerable interest to determine:

  • How they respond to stress, e.g., prolonged (time-course) propofol exposure.

  • How propofol exposure (dose response) directs/signals these cells to undergo apoptosis or necrosis.

  • How their proliferation rate is affected by short- or prolonged anesthetic exposure.

  • How their fate, e.g., differentiation from progenitor cells to neurons or glial cells, is affected.

  • How the anti-oxidant agents affect anesthetic-induced neurotoxicity.

  • Clinically relevant protective strategies against anesthetic-induced developmental neural damage.


Embryonic neural stem cells potential anesthetic induced neurotoxicity

LDH Release

(Propofol; 24-hour Exposure)


Embryonic neural stem cells potential anesthetic induced neurotoxicity

MTT-Assay

(Propofol; 24-hour Exposure)

*

*

a


Embryonic neural stem cells potential anesthetic induced neurotoxicity

A

3-hour Exposure

6-hour Exposure

B

Control Propofol (10 µM) propofol (50 µM) Propofol (100 µM)


Embryonic neural stem cells potential anesthetic induced neurotoxicity

Propofol Exposure

(24-hour)

A

B

Control

Propofol (50 µM; 24 hours)


Embryonic neural stem cells potential anesthetic induced neurotoxicity

TUNEL-Assay

A

B

Control

Propofol (50 µM)


Embryonic neural stem cells potential anesthetic induced neurotoxicity

Scatter Plot (Sorting)

Cellometer Vision

Control

Propofol (50 µM; 24 hours)


Embryonic neural stem cells potential anesthetic induced neurotoxicity

EdU-DAPPI Staining

Control

Propofol (50 µM; 24 hrs)

A

B

EdU

C

D

DAPPI

E

F

Edu-DAPPI


Embryonic neural stem cells potential anesthetic induced neurotoxicity

Neural Stem Cell Proliferation

(Propofol; 24-hour Exposure)

*


Embryonic neural stem cells potential anesthetic induced neurotoxicity

*

Control Pro 3 hours Pro 6 hours Pro 24 hours

Pro = Propofol (50 µM)


Embryonic neural stem cells potential anesthetic induced neurotoxicity

Assessment of Mitochondrial Membrane Potential

Control

Propofol-exposed


Embryonic neural stem cells potential anesthetic induced neurotoxicity

8-oxo dG ELISA-Assay

(Propofol; 24-hour Exposure)


Embryonic neural stem cells potential anesthetic induced neurotoxicity

*

Control Propofol (50µM) Propofol+L-Ca L-Ca alone

L-Ca = Acetyl-L-Carnitine (10 µM)


Embryonic neural stem cells potential anesthetic induced neurotoxicity

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.


Embryonic neural stem cells potential anesthetic induced neurotoxicity

Propofol

Excitatory-R

Ca2+

Anions

Cations

Neural Stem Cell

GABA-R

ROS

GABA-R

Disturbed

Cell Cycle

Anion

Cation

unbalance

Anions

Cations

cell death

Mitocondrion

G1

L-Carnitine

M

cytochrome c

G2

S

DNA Fragmentation

Chromatin

Condensation

caspases

DFF40/CAD


Embryonic neural stem cells potential anesthetic induced neurotoxicity

ACKNOWLEDGEMENTS

NCTR/FDA

CDER/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


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