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