Transcript Progesterone treated 72hr TBI Mice

Progesterone Modulates the
Phosphorylation of Akt in a Closed
Skull Traumatic Brain Injury Model
Justin Garling, MSII, Lora Watts, PhD, Shane Sprague, BS, David F. Jimenez, MD, FACS,
Murat Digicaylioglu, MD, PhD
The University of Texas Health Science Center at San Antonio
Introduction
•
Results
Traumatic Brain Injury (TBI) affects
nearly 1.7 million people in the United
States each year, but not all head
injuries result in TBI.2
There are currently no FDA approved
drugs on the market to treat TBI
Progesterone is a hydrophobic steroid
hormone that has been shown in
recent studies to exhibit
neuroprotective effects.
This study aims to determine if
progesterone is involved in the
regulation of Akt via the Serine 473
and Threonine 308 phosphorylation
sites.
•
•
•
Progesterone Treated Astrocyte Culture
(Dose response curves)
Progesterone
concentrations:
PGR
Merged
10nM
20nM 40nM 100nM
1μM
20μM
P-Akt (S473)
Total Akt
Actin
Progesterone treated 72hr TBI Mice
TBI w/P4
Ctx
H
TBI w/P4
Sham w/P4
Ctx
Ctx
H
Sham w/V
H
Ctx
H
P-Akt (T308)
Total Akt
Figure 1: The progesterone treated astrocyte cultures were incubated for
30min. The vehicle (V) used in all experiments was 2-hydroxypropyl-βcyclodextrin. The right cortex (Ctx) and hippocampus (H) were isolated in the
progesterone treated 72hr TBI mice
Hippocampus of TBI mice
Sham
24hr TBI
72hr TBI
40X
20X
GFAP
1nM
V
P-Akt (T308)
Expression of Progesterone Receptor
DAPI
C
Merged
PGR
100X
DAPI
Figure 2: Progesterone receptor in Astrocyte culture and mouse
cortex. DAPI=blue, nuclei. PGR (progesterone receptor)= green.
GFAP (Glial Fibrillary Acidic Protein)= red, and is a structural
protein of astrocytes.
Figure 3: Nissl Staining. Nissl stains rough endoplasmic reticulum. All Nissl stainings
show the hippocampus of the impacted side of C57Black/6 mice. None of the mice
shown were treated with progesterone.
Conclusions
Progesterone (100 nM) treated astrocyte
culture:
Progesterone
S473
• Increased phosphorylation of Akt at Thr308
• Decreased phosphorylation of Akt at
Ser473
• Small decrease in total Akt
P
Akt
Growth
P
Apoptosis
Edema
TBI mice treated with Progesterone:
T308
• Significantly increased phosphorylation of
Akt at Thr308 site in the hippocampus
compared to sham (72 hours post-TBI)
Inflammation
Significance of Researching Progesterone:
• To identify the active groups on the
progesterone molecule.
• Progesterone is a large molecule with
solubility and blood brain barrier
penetration problems.
• To find a progesterone mimetic
Cell
Viability
Future Directions
Methods
Mouse Traumatic Brain
Injury (TBI) model:
A 5mm disc is placed over
the right side of the exposed
skull between the bregma
and lambda sutures. Mice
were impacted using a
pneumatic impactor which
fires at a rate of 4.5m/s
striking the disc depressing
the skull 2mm.
Progesterone Treatment:
•
Time-response curves at the
progesterone concentrations which
produced the most significant
changes in the dose-response curves
(100nM)
•
Oxygen Glucose Deprivation (OGD) in
astrocyte and cortical neuron cultures
•
P-mTOR pathway relation to
progesterone and Akt
•
Intranasal progesterone treatment
Mice were treated with progesterone by injection postTBI at 1hr (i.p.), 6hr (s.c.), 24hr (s.c.), 48hr (s.c.), and
then sacrificed at 72hrs. (Note: i.p.=intraperitoneal
s.c.= subcutaneous)
Progesterone
References
1.
2.
3.
Brinton, R., Thompson, R. & Foy, M. Progesterone
Receptors: Form and Function in Brain. Neuroendocrinology
29, 313-339 (2008).
Control, C.f.D. CDC - Injury - Traumatic Brain
Injury(Atlanta, 2010).
Cutler, S., Cekic, M., Miller, D. & Stein, D. Progesterone
Improves Acute Recovery after Traumatic Brain Injury in the
Aged Rat. Journal of Neurotrauma, 1475-1486 (2007).
4.
5.
CW, G., SW, H. & DG, S. Behavioral effects and anatomic
correlates after brain injury: a progesterone dose-response
study. Pharmacology, Biochemistry, and Behavior, 231-242
(2003).
Wright, D., Bauer, M., Hoffman, S. & Stein, D. Serum
Progesterone Levels Correlate with Decreased Cerebral
Edema after Traumatic Brain Injury in Male Rats. Journal of
Neurotrauma 18 (2001).
This work was supported by the Department of Neurosurgery