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Preliminary Results of Optic Nerve Crush Damage in the 13 Lined Ground Squirrel
Ryan
1
Bures ,
Ben
1
Sajdak ,
Grant
1
O’Connell ,
Cassandra
1
Piper ,
David
2,3
Henson
and Dana
1
Merriman
1.UW Oshkosh Biology & Microbiology, University of Wisconsin Oshkosh, Oshkosh, WI
2. Hanover College 3. REU Student at Wisconsin Oshkosh
The UW Oshkosh McNair Scholars Program is 100% funded through a TRIO grant from the
United States Department of Education PR/Award Number P217A070188. For 2012/2013, the
UW Oshkosh McNair Scholars Program will receive $213,000 each year in federal funds.
Purpose
Methods
Whenever the central nervous system (CNS) of humans, monkeys, cats,
rabbits, rats, or mice is damaged, neurons die and two other cell types,
microglia and astrocytes, undergo activation to make emergency repairs to
the tissue. Taken together, their activation is known as “reactive gliosis”.
Upon insult to the CNS, microglia undergo a shift from a resting state to an
activated state and proliferate. In this activated state, the microglia cells
become phagocytes sequestering neuronal debris. They also release IL-2 that
signals astrocytes to activate new gene expression programs involving motility
and cytoskeletal reorganization. The reactive astrocytes form a glial scar that
reinforces the blood-brain barrier (damaged by the initial insult) and fills the
space vacated by dead neurons. Hallmarks of reactive gliosis include
significant upregulation of the cytoplasmic intermediate filament proteins,
vimentin and glial fibrillary acidic protein (GFAP), within astrocytes, as well as
the extracellular deposition of chondroitin sulfate proteoglycans (CSPGs) in new
basement membrane. These damage responses accomplish tissue repair, but
inhibit the ability of surviving neurons or therapies such as stem cells to repair
lost neuron circuitry.
In very limited studies to date, the ground squirrel CNS shows a uniquely
muted microglial and glial response to damage, with minimal reaction to neuron
death. As such, the squirrel’s version of reactive gliosis could represent the
best-case scenario for repairing neuron circuitry in human patients suffering
CNS damage due to trauma or disease. Whereas cell signal pathways have
been partially elucidated for full-version reactive gliosis in typical rodent models
(rat and mouse), nothing is known of the attenuated pathways in ground
squirrel. The long-term objective in our lab is to determine the differences in
signal pathways that account for the different outcomes, with possible
translational value to human medicine.
The goal of this pilot study was to achieve proficiency in (a) a new-to-us
damage model, known as “optic nerve crush” (ONC), and (b) initial
immunolocalization of three key damage response markers: GFAP and vimentin
expression and CSPG deposition. With these methods in hand, we will be
positioned to compare and contrast signal pathways.
Optic nerve crush damages TWO tissues: the nerve itself, which is devoid of
neuronal cell nuclei (but is populated by nuclei of astrocytes and microglia); and
retrograde apoptotic neuron death in the retina (Fig. 1). Therefore, we collected
both optic nerve and retina tissue in our experiments.
Long-Evans rats, which undergo the full reactive gliosis program after ONC,
served as our positive control species. One eye from each animal received
sham treatment as a negative control. A time-series of survival was arranged to
attempt to “catch” the canonical chain of events after nerve crush, which is
thought to be:
• Microglial activation (and onset of neuron apoptosis)
• Vimentin upregulation
• GFAP upregulation
• CSPG deposition
In both rats and squirrels, we used (Fig. 2):
• On-section immunolabeling using anti-vimentin, anti-GFAP, and antiCSPG to assess reactive gliosis.
• Electron microscopy analysis of morphology changes.
All animal procedures complied with the UW Oshkosh IACUC approval and all
federal animal welfare guidelines. Thirteen-lined ground squirrels (Ictidomys
tridecemlineatus) were obtained from the UW Oshkosh breeding colony. Rats (Rattus
norvegicus) were obtained from Charles Rivers.
McNair Scholars Program: Award Number P217A070188, National Science Foundation Research Experience for
Undergraduates, 2012
Sources and Acknowledgements
Tsung-I, Kuan-Ming F, Shun-Fen T. Role of Ciliary Neurotrophic Factors in Microglia Phagocytosis. Neurochemical
Research. January 2009; 34(1):109-117
Rolls A, Shechter R, Schwartz M. The bright side of the glial scar in CNS repair. Nature Reviews Neuroscience. March
2009;10(3):235-241
Linberg K, Sakai T, Lewis G, Fisher S. Experimental retinal detachment in the cone-dominant ground squirrel retina;
Morphology and basic immunocytochemistry. Visual Neuroscience. 2002; 19 603-619
Berlin Memorial Hospital for help embedding and sectioning tissue
Fix
4% Paraformaldehyde
Optic nerve
crush site –
local glial
response
Fix
2% Paraformaldehyde
2% Glutaraldehyde
Embed
Embed
Optic nerve with
axons, astrocytes,
& microglia
Section
Section
Immunolabel for
GFAP, Vim, CSPG,
and IBA 1
Figure 1.
Representation
of retina and
optic nerve postcrush, not
representative of
scale or
concentration
Investigate
Morphology by
Electron Microscopy
(data not shown)
Arrow Key:
Procedures completed
Preliminary data obtained, troubleshooting
ongoing
Cross-section Immunolabeled Optic Nerve
Vimentin
24 Hour Post-Crush
GFAP
Figure 3.
Differential expression of
proteins associated with
glial scarring between
the rat and squirrel postONC (400x). As expected,
expression of vimentin and
GFAP was higher in the rat
than in the squirrel at 24
hours post-injury. These
two markers clearly label
hypertrophic astrocytic
processes at this time
period. The extracellular
macromolecules CSPGs
similarly show a higher
deposition in the rat
compared to the squirrel at
24 hours. While data
concerning baseline
expression of these
proteins is not yet
processed, these
preliminary results suggest
a more robust glial
response in the damaged
rat optic nerve.
24 Hour Post-Crush
Ganglion cell nuclei
2 Weeks Post-Crush
Retinas and
Optic Nerves
Figure 2.
Flow Chart of
methods
24 Hour Post-Crush
Retina with neurons
and astrocytes scattered neuron death
and glial response
2 Weeks Post-Crush
Left Eye: ONC injury
Right Eye: Sham
CSPGs
Funding Support
GFAP Immunolabeled Optic Nerve
Figure 4. GFAP immunoreactivity at 24 hours and 2 weeks post crush.
Damaged rat optic nerve shows more GFAP labeling at 24 hours than the squirrel
optic nerve (A, B). Compared to 24 hr, GFAP expression at 2 weeks is higher in
squirrel astrocytes (C), but they do not display the severe hypertrophic gliosis seen
in the rat at 2 weeks (D).
Conclusions & Future Work
Vimentin, CSPG, and especially GFAP expression in the damaged optic nerve of
the rat is consistently higher than that seen in damaged squirrel optic nerve. This was
most evident at 2 weeks post-crush (Fig. 4C, D) when glial scars are well-developed in
typical species such as the rat.
These data corroborate our earlier demonstration that ground squirrel retina
exhibits attenuated reactive gliosis following detachment (data available from presenter).
We continue to troubleshoot procedures for electron microscopy and will soon begin to
analyze our banked tissue (optic nerves and retina) for apoptotic cell death and
microglial activation.