1. 0
20
40
60
80
100
120
No Drug Buprenex Only
Number of parvalbumin positive cells
t-Test: Two -Sample Assuming Equal Variances
Buprenex Only No Drug
Mean 98.66666667 58.83333
Variance 227.5833333 30.58333
Observations 3 3
Pooled Variance 129.0833333
Hypothesized Mean Difference 0
df 4
t Stat 4.293952088
P(T<=t) one-tail 0.006353153
t Critical one-tail 2.131846782
P(T<=t) two-tail 0.012706306
t Critical two-tail 2.776445105
83
94
88.5
62
66
64
Dr. William D. Eldred Department of Biology
Examining the Changes in Calcium Binding Protein Expression
Following Blast Induced Brain Injury Using C57BL/6 Mice
Eric Schmidt, Shama Patel
ABSTRACT
EXPERIMENTAL DESIGN
GOALS
We would like to specially thank Gloria DeWalt, Dr. Todd Blute and Dr. William D. Eldred for
their guidance and knowledge throughout our research study. We would also like to thank the
other research students in the Eldred lab that helped us over the summer: Bryan Duong, Biraaj
Mahajan and Sara Mansuri. Funding was generously provided by BU UROP.
Traumatic brain injury (TBI) has been linked to
alterations in calcium homeostasis. TBI can lead to an
influx of calcium ions following injury to the brain that
can be coupled to subsequent cell death through
excitotoxicity.
One of our research goals was to analyze the visual
cortex (V1) and the retrosplenial cortex (RSC) in the
brains of C57BL/6 mice since perturbations in vision and
memory are prevalent symptoms following blast induced
TBI in humans. In order to investigate this goal, a rodent
blast apparatus was used to deliver a nonlethal
overpressure blast to restrained mice in the presence or
absence of the analgesic Buprenex. Following a 48 hour
recovery period, animals were sacrificed, then
immunocytochemistry and confocal microscopy were
utilized to characterize the differences between blasted
mice with and without anesthetics.
The calcium-binding protein parvalbumin was chosen
based on it’s prevalence and ability to identify non-
overlapping neuronal populations in the cortex. Using
antibodies directed against the parvalbumin, we quantified
the number of neuronal cell bodies stained in the cortex.
Our results showed a statistically significant difference
between blasted mice with and without Buprenex. We
conclude that using Buprenex in TBI research may
significantly confound the results.
Figure 1. Cranial
Only Blast Injury
Apparatus (COBIA)
developed by Kuehn
et. al.
In this rodent model, a
nonlethal blast
overpressure is
delivered to the head of
live awake mice using
a 0.22 caliber
smokeless powder
blank cartridge. The
blast is delivered
vertically downwards
directly onto just the
head of the retrained
mouse.
METHODS
Blast Injury Procedure
Our model focuses on blast induced trauma using a Cranial Only Blast
Injury Apparatus (COBIA) developed by Kuehn et al., 2011. The mice
that received Buprenex (0.1mg/kg) were injected at least 10 minutes
prior to blast. A paper cone was used to position the head of mice during
blast. The method and procedures developed adhere to the Guide for
Care and Use of Laboratory Animals.
Tissue Preparation
To prepare the tissue, the mice were anesthetized with isofluorane gas
after a 48 hour survival time and then perfused with fixative and
decapitated. The isolated brain tissue was cryoprotected by placing it in
sucrose solutions of increasing concentrations from 5-30% and left
overnight at 4°C. The tissue was then immersed in embedding media,
frozen, and sliced into 30 μm-thick sections using a microtome. The
tissue was then mounted onto Superfrost/Plus slides for
immunocytochemistry.
Immunocytochemistry
The sections on slides were then sequentially treated with normal
blocking serum, followed by primary and secondary antibodies diluted
in 0.3% Triton X100 and phosphate buffer. The primary antibody was
directed against the calcium binding protein parvalbumin (Novus,
1:5000). The slides were cover slipped with VectaShield.
Image Analysis
The fluorescent labeling was visualized using a Fluoview 10i confocal
microscope. The images were subsequently analyzed using Image J
software to generate inverted images such that signal appeared black on
a white background. The cell counting plugin in Image J was used to
quantify the number of labeled cells.
• To examine the differences in visual cortex and
RSC between sham and blast mice by examining
the calcium binding protein parvalbumin.
• To elucidate the effects of the analgesic Buprenex
anesthetic on TBI.
• To identify which cell signaling pathways are
involved in blast pathology in order to provide a
basis for future research about prevention or
treatment of traumatic brain injuries.
QUANTITATIVE METHODS
FUTURE DIRECTIONS
• In future research, we will carry out similar studies analyzing calcium binding
proteins in the mouse retina and see if significant similarities exist. If so, we may
be able to detect TBI in the brain by analyzing the effects in retina.
• We will do further immunocytochemical studies analyzing other calcium binding
proteins to determine how wide spread the effects are.
• We are also focusing on other brain regions such as the dentate gyrus within the
hippocampus and other corteces.
STATISTICAL RESULTS
CONCLUSIONS
BIBLIOGRAPHY
Figure 2. Parvalbumin
antibody labeling in the
retrosplenial cortex.
Effects of Buprenex on sham and
blasted mice (677, 676, 621,
618). Buprenex decreased the
number of labeled cells in sham
mice and it increased the number
of parvalbumin labeled cells in
the blasted mice.
Figure 3. Statistical
analysis of the effects of
Buprenex on
Parvalbumin levels in
blasted visual cortex.
Results from two different
sections from the same
mouse are shown
vertically. The red
numbers indicate the
number of labeled cells in
each image. The blue
numbers indicate average
number from these two
sections. The statistics are
taken from two different
sections taken from three
different mice.
• Levels of paralbumin are statistically changed in response to blast TBI.
• Buprenex decreases the number of labeled cells in sham mice and it increases the
number of parvalbumin labeled cells in the RSC in blasted mice.
• Buprenex significantly changes the effects of blast in V1 of visual cortex
• The effects of drugs may confound the interpretation of many previous studies of
TBI effects in animal model systems.
Sham Buprenex only Blast Buprenex only
Sham No Drug Blast No Drug
Parvalbumin
Sham no drug Blast no drug
Avg. # of cells 209 cells 87 cells
Average
labeling
intensity
Mean 74
Mean 62
Sham +Bup Blast + Bup
Avg. # of cells 91 cells 139 cells
Average
labeling
intensity
Mean 61
Mean 59
Buprenex Blast No Drug Blast
Reed Kuehn, Philippe F. Simard, Ian Driscoll, Kaspar Keledjian, Svetlana Ivanova, Cigdem
Tosun, Alicia Williams, Grant Bochicchio, Volodymyr Gerzanich, and J. Marc Simard
Rodent Model of Direct Cranial Blast Injury, Journal of Neurotrauma 28:2155–2169
(2011)
Quantification in Image J
ACKNOWLEDGEMENTS