1. Evaluating Time-Dependant Effects of
Maximal Electroshock on GFAP and
NeuN Immunostaining in Rats
TERESA DUARTE
ANTICONVULSANT DRUG DEVELOPMENT (ADD) PROGRAM
Westminster College, Junior

2. INTRODUCTION
Anticonvulsant Drug Development Program
• Uses animal and in vitro models of seizures and epilepsy to
identify new antiepileptic agents.
Why is this important?
•Epilepsy
1 in 26
Over 50 million people worldwide
Neurological disorder
Repeated seizures
What is a seizure?
• Disturbed brain activity
• Behavioral/Attention changes
•Treatments are available, but more are needed

3. MES Testing
Maximal Electroshock (MES) Model of Seizures
• Common model for routine screenings
• Used to identify potential anti-seizure drugs
5% of male Sprague-Dawley rats do not respond to MES
• Do not have a tonic-clonic seizure with hind-leg extension
• “Responders” vs. “non-responders” (R vs. NR)
Male Sprague-Dawley rats were stimulated via MES (50 Hz)
• Animals euthanized at 5, 60 and 120 min after MES
• Tissue sections used are from this test
• Different neuroanatomy in NR vs R

4. Analysis of the Rat Hippocampus
Dentate Gyrus
Hippocampus

5. Analysis of Protein Expression by
Immunohistochemistry
Proteins of Interest
• NeuN  Neurons
• GFAP  Astrocytes  Type of Glial Cell
• DAPI  Marker for nuclei of all cells
Images were Captured and Analyzed
• Fluorescence microscopy
• Densitometric analysis
• Quantification of total signal

6. EXPERIMENTAL
GFAP/NeuN Protocol
Brain tissue is sliced and mounted onto slides, stained
with NeuN and GFAP antibodies, followed by DAPI.
DAPI Stain NeuN Stain GFAP Stain
100 um

7. Composite Image
DAPI – Yellow
NeuN – Cyan
GFAP - Magenta

8. DAPI Stain
(Nuclei)
NeuN Stain
(Neurons)
GFAP Stain
(Astrocytes)
Dentate (In Hippocampus) Imaging
100 um

9. COMPOSITE IMAGE
Purple – GFAP
Yellow – NeuN
Green -- DAPI
10 um

10. GFAP Expression in NR vs R
• two time points
•Sample size was small
R
e
s
p
o
n
d
e
r
N
o
n
-R
e
s
p
o
n
d
e
r
0
2 0
4 0
6 0
8 0
G F A P 6 0 M P e rc e n t A re a
6 0 M in u te s A fter M E S
PercentAreawithSignal
R
e
s
p
o
n
d
e
r
N
o
n
-R
e
s
p
o
n
d
e
rs
0
2 0
4 0
6 0
8 0
G F A P 1 2 0 M P e rc e n t A re a
1 2 0 M in u te s a fte r M E S
PercentAreawithSignal
R
e
s
p
o
n
d
e
r
N
o
n
-R
e
s
p
o
n
d
e
r
0
2 0
4 0
6 0
8 0
1 0 0
G F A P 6 0 M M e a n G ra y
6 0 M in u te s A fter M E S
MeanGrayValueofLabeledPixels
R
e
s
p
o
n
d
e
r
N
o
n
-R
e
s
p
o
n
d
e
r
0
2 0
4 0
6 0
8 0
1 0 0
G F A P 1 2 0 M M e a n G ra y
1 2 0 M in u te s a fte r M E S
MeanGrayValueofLabeledPixels

11. NeuN Expression in NR vs R
R
e
s
p
o
n
d
e
r
N
o
n
-R
e
s
p
o
n
d
e
r
0
2 0
4 0
6 0
8 0
N e u N 6 0 M P e rc e n t A re a
6 0 M in u te s A fte r M E S
PercentAreawithSignal
R
e
s
p
o
n
d
e
r
N
o
n
-R
e
s
p
o
n
d
e
r
0
2 0
4 0
6 0
8 0
N e u N 1 2 0 M P e rc e n t A re a
1 2 0 M in u te s a fte r M E S
PercentAreawithSignal
R
e
s
p
o
n
d
e
r
N
o
n
-R
e
s
p
o
n
d
e
r
0
5
1 0
1 5
N e u N 6 0 M M e a n G ra y
6 0 M in u te s A fte r M E S
MeanGrayValueofLabeledPixels
R
e
s
p
o
n
d
e
r
N
o
n
-R
e
s
p
o
n
d
e
r
0
5
1 0
1 5
N e u N 1 2 0 M M e a n G ra y
1 2 0 M in u te s a fte r M E S
MeanGrayValueofLabeledPixels
*
Significant results in NeuN
120 M Mean Gray value
All these data suggest a
potential difference in
neuronal density between
NR and R

12. 5
M
in
u
te
s
6
0
M
in
u
te
s
1
2
0
M
in
u
te
s
0
2 0
4 0
6 0
G F A P R P e rc e n t A re a
T im e A fte r M E S
PercentAreawithSignal
5
M
in
u
te
s
6
0
M
in
u
te
s
1
2
0
M
in
u
te
s
0
2 0
4 0
6 0
8 0
1 0 0
G F A P R M e a n G ra y
T im e A fte r M E S
MeanGrayValueofLabeledPixels
5
M
in
u
te
s
6
0
M
in
u
te
s
1
2
0
M
in
u
te
s
0
2 0
4 0
6 0
8 0
N e u N R P e rc e n t A re a
T im e A fte r M E S
PercentAreawithSignal
5
M
in
u
te
s
6
0
M
in
u
te
s
1
2
0
M
in
u
te
s
0
5
1 0
1 5
N e u N R M e a n G ra y
T im e A fte r M E S
MeanGrayValueofLabeledPixels
No significant results.
Does time after MES affect NeuN and GFAP protein expression?

13. CONCLUSION
 The MES Test is a very useful model of seizures to identify new drugs for the
treatment of epilepsy.
 The development of new antiepileptic drugs is imperative towards ameliorating
epilepsy.
 Its important to have well validated and well understood animal models to best
develop drugs.
 This project may suggest that there are neuranatomical differences in the
expression of cell specific proteins between MES R and NR.
Future Direction
Increase sample size for R and NR
Evaluate other cell specific markers found in the brain (i.e microglia)

14. Dr. Melissa Haliski
Dr. Steve H. White
ADD Program Laboratory Staff
Graduate Preparation Institute – The University of Utah

16. Identification
MES
32 mA 6 Hz test
Corneal kindled mouse
Bursting hippocampal slice
Initial Differentiation
44 mA 6 Hz test
LTG-resistant kindled rat
iv PTZ seizure threshold
Behavioral toxicity tests Quantification
(time- and dose-response)
Stop
testing
inactive
Full Differentiation
Post-SE epileptic rat/mouse
Pediatric seizure or epilepsy
model
In vitro neuroprotection
One or more non-seizure
models
active
active
most promising
compounds
advance
University of Utah - Anticonvulsant Drug
Development Program
Screening of Investigational Compounds for the Treatment of
Epilepsy