Project Title: Dye wanted: dead, alive, or zombies?

The utility of a novel fluorescent dye to study cell death

in vivo
Rationale: Rat models of spinal cord injury (SCI) are effective tools to study traumatic injury to the central
nervous system. For example, this laboratory uses a well-characterized weight-drop model of SCI to study the
molecular mechanisms of secondary injury, which in many cases leads to neuronal injury/death. Two distinct
subtypes of cell death present in the injured spinal cord: (i) apoptotic and (ii) necrotic. Necrotic cell death is
traditionally thought to result from complete energy depletion, which renders the cell unable to regulate its
internal environment and results in the activation of various autolytic processes. In contrast, apoptotic cell
death, as the pre-programmed mode of cell demise, is subject to energy expenditure regulated by homeostatic
mechanisms. In apoptotic cells, the conservation of the plasmalemma is an energy-demanding process that
results in minimal immune response. However, as the depletion of energy in necrotic cells renders a lack of
plasmalemma integrity, the exposure of the cytoplasmic content to the extracellular space renders a massive
inflammatory response. The catastrophic downstream effects of unregulated necrotic cell death therefore
underlie a far greater pathophysiological response than those of apoptotic cell death. The progression of
necrotic cell death, as a reflection of a gradual loss in membrane integrity and cell swelling, is therefore able to
be classified on basis of severity. The most common way to evaluate necrotic cell death in SCI, is to label
necrotic cells with Propidium Iodide (PI). Propidium Iodide, a membrane impermeant compound that fluoresces
once bound to nucleic acids specifically enters necrotic cells due to their compromised plasmalemma.
However, strong PI fluorescence is also observed in cells that have not progressed far along the cascade of
events that culminates in necrosis. For studies that require discrimination between necrotic stages, this
property of PI invites question. In the weight-drop model of SCI, cells that received comparatively less
mechanical trauma, and were positive for PI labeling, gave off a signal nearly identical to PI positive cells that
received the most impact. Although accurately displaying necrosis in affected cells, this signal strength does
not accurately represent the cellular progression of necrosis present in spinal cord injury. Thus, PI is a useful
tool, but it is insufficient for nuanced study of necrotic cell death, as it is unable to accurately distinguish the
stages of this process. Additional tools used to label necrotic cell death in the weight-drop rat model of spinal
cord injury are needed, as the accurate representation of neuronal death requires that
The line of fluorescent Zombie dyes produced by BioLegend introduces a new perspective to the
microscopic analysis of necrotic cell death as a whole. Zombie dye fluoresces when bound to the primary
amine groups of proteins, and therefore labels membrane proteins in cells that do not have a compromised
membrane. However, in cells with compromised membrane integrity, the membrane-impermeant dye is able to
label intracellular proteins. Thus, necrotic cells with compromised membrane integrity will fluoresce to a much
greater degree than cells with intact membranes. I believe that that this mechanism of action allows for cells
with greater degrees of membrane damage to fluoresce to a greater extent than those with lesser membrane
permeability, effectively demonstrating the varying severity of necrotic cell death. Commonly used in flow
cytometry, this line of dyes has never been utilized in microscopy. This proposal aims to determine
whether Zombie dye is an effective tool to study the stages of necrotic cell death in rat models of SCI. This
instrument will be of immense importance to our research with rat models of CNS injury, as it might allow for a
much more accurate depiction of the nature of necrotic cell death. The experiments detailed below are
feasible; I have had nearly a year of experience of being mentored in spinal cord injury research and
performing advanced immunofluorescent protocols. I will be working closely with Dr. J Marc Simard; the
Principal Investigator of the Neurosurgery Research Labs, who has many years of experience working with
spinal cord injury project and the immunofluorescent protocols associated with them. Dr. Simard will be
assisting with the experimental design of this project, and will supervise its execution. The successful
completion of this proposal will not only effectively increase the breadth and depth of my knowledge in
neurosurgical research as a whole, but more importantly bring a novel method of characterizing necrotic cell

death in pivotal models of spinal cord injury to my home laboratory. For these aforementioned reasons, this
project is well suited to be supported by a SMART award.
Hypothesis: Zombie dye is superior to Propidium Iodide to demonstrate stages of necrotic cell death in the
weight-drop model of spinal cord injury in rats.
Specific Aim: Demonstrate whether Zombie dye is superior to PI in the demonstration of stages of necrotic
cell death.
Research Approach: Rats (n=3) will be injected with both Propidium Iodide and Zombie dye through venous
tail catheterization 15 minutes before the application of weight-drop induced spinal cord injury. After euthanasia
10 minutes post-SCI with sodium pentobarbital, rats will be perfused transcardially with saline, followed by 4%
Paraformaldehyde (PFA). Spinal cords will then be immersion post-fixed in 4% PFA and cryoprotected in 30%
sucrose solution. 10 um coronal cryosections will be sectioned using a cryostat. Sections will be examined with
excitation/emission microscopy. Each dye possesses a separate excitation and emission spectra. Propidium
Iodide excites and emits at 540 nm and 620nm, and Zombie Violet at 385nm and 420nm. In order to
demonstrate varying stages of cell death, PI positive cells exhibiting Zombie dye labeling will be referenced to
PI negative cells exhibiting Zombie dye labeling. PI negative cells in the side contralateral to the injury will be
used as a baseline value for comparing higher degrees of fluorescence in cells in the damaged region. The
hypothesis will supported with the demonstration that PI positive cells further from the impact site will possess
a lesser percentage difference in fluorescence than PI positive cells closer to the impact site.
Impact: This study seeks to broaden the methods available to this laboratory, and many others investigating
the pathophysiology of the central nervous system. In the Simard lab, the mechanisms of secondary injury we
investigate involve accidental necrotic cell death mediated by the Sur1-Trpm4 channel. PI staining in the
demonstration of necrotic injury has been used in multiple models of in vivo neurological injury ranging from
stroke, subarachnoid hemorrhage, traumatic brain injury, and spinal cord injury. Usage of PI in all of these
models fails to demonstrate a graded nature of necrotic cell death highly applicable to the molecular
mechanisms that mediate secondary injury. The outcomes of this experiment will introduce a novel fluorescent
dye that will be used to characterize the nature of cell death in vivo models of neurological injury. By
demonstrating the usage of the dye in this model, not only will a new depth of insight be gained into the rat
model of SCI, but would be the first project utilizing Zombie dye in microscopy. The implementation of a
novel dye in the immunofluorescent characterization of necrotic cell death will bring an improved model of
neurological injury to both my home laboratory, as well as those in the neurosurgery community.
Reagents: I have selected BioLegend as my preferred sponsor. Propidium Iodide and the Zombie Violet
Fixable Viability Kit are requested in order to label necrotic cells in the weight-drop model of spinal cord injury
in rats.
Investigator Information:
1. Primary Investigator of Laboratory: J. Marc Simard, M.D., Ph.D.
2. Email address: msimard@smail.umaryland.edu
3. Address: 10 S. Pine St., MSTF Rm 634B, Baltimore, MD 21201
Student conducting the project: Bradley Smith
Educational status: High School student working under the direction of Dr. J Marc Simard
Email address: smith.brad.richard@gmail.com
Lab Address: 10 S. Pine St., MSTF Rm 634B, Baltimore, MD 21201

Mentor assisting with new technology or technique: Dr. J Marc Simard, M.D., Ph.D.
Email address: msimard@smail.umaryland.edu