The document summarizes an experiment that investigated the effects of granulocyte colony stimulating factor (GCSF) on rats that experienced stroke. Rats were divided into groups that either received or did not receive GCSF treatment after experiencing induced stroke. Western blot assays were used to analyze and compare the molecular expression of caspase 12 and glucose regulated protein 78 (GRP78), which are indicators of cell death, between the treated and untreated groups in the core and penumbra regions of the brain. The results found decreased expression of both proteins in the core of treated rats but increased expression in the penumbra, though the results were not statistically significant. Future research with larger sample sizes is needed to better understand the effects.

1. THE EFFECTS OF
GRANULOCYTE
COLONY
STIMULATING
FACTOR ON
STROKE INDUCED
RATS
Matthew Busel
Pine Crest School
Work conducted at Florida Atlantic
University

2. INTRODUCTION: GCSF
 Granulocyte Colony Stimulating Factor (GCSF) is a growth factor
and cytokine that stimulates the proliferation and mobilization of
hematopoietic stem cells
 Its actions are mediated by binding to specific GCSF receptors
(GCSFRs)
 Although its main clinical use has been to counteract neutropenia,
recent studies have reported that GCSFRs are located on the
brain leading to neuroprotective effects of the drug
 By decreasing the rate of neuronal death many central nervous
system (CNS) disorders may be counteracted against

3. INTRODUCTION: CEREBRAL ISCHEMIA
 Focal cerebral ischemia (stroke) is a CNS disorder in which the
cerebral blood vessels are blocked, resulting in an inadequate
blood flow to the brain and subsequent oxygen (hypoxic) –
glucose deprived environment
 In this state the brain cannot meet metabolic demands and
neuronal death will eventually be initiated via apoptosis
 The resulting ischemic infarct (area of dead cell tissue) includes a
central core (an area of severe ischemia) and a surrounding
penumbra, which is vulnerable if the cell death is not arrested
White = core
Red = penumbra

4. INTRODUCTION: APOPTOTIC
PATHWAYS
 The endoplasmic reticulum (ER) is responsible for the proper folding
and sorting of proteins
 During an ischemic insult the ER becomes stressed and will try to
restore normal function by deactivating protein translation signaling
pathways while activating other pathways that increase the
production of chaperone molecules that facilitates protein folding
 If it is unsuccessful in rectifying its stressed state and restoring
normal function, the ER will initiate an apoptotic cascade
 Caspase 12 is an ER-membrane associated protein and one of the
initiators of the caspase-mediated apoptotic pathway
 Glucose regulated protein 78 (GRP78) is a chaperon molecule
associated with the ER

5. INTRODUCTION: EXPERIMENT
 The molecular expression of GRP78 and Caspase 12 were
compared in GCSF treated and untreated rats
 Both GRP78 and Caspase 12 are excellent indicators of cell
death

6. HYPOTHESIS
 If the molecular expression of GRP78 and Caspase 12 are
measured in GCSF treated and untreated rats after focal cerebral
ischemia, treated rats will have lower expression of the selected
indicators and therefore lower cell death in both the core and
penumbra of the brain

7. METHODS: WESTERN BLOT
 Frozen homogenates/lysates of protein samples (from two rats to
increase diversity with sample population) were used to perform
western blot assays
 Briefly, protein homogenates/lysates were boiled for 5 minutes and
centrifuged
 Each lane was loaded with 50 μg of protein and subjected to sodium
dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) on
12% gels and then electrophoretically transferred to nitrocellulose
membranes
 Once protein transfers were completed, the nitrocellulose
membranes were blocked in 5% nonfat dry milk in TBST (20 mmol/L
Tris base, pH 7.6, 137 mmol/L NaCl, and 0.05% Tween 20) for 1 hour
at room temperature and rinsed in TBS-T (50 mM Tris, 170 mM NaCl,
0.2% [vol/vol] Tween 20; pH 7.5)

8. METHODS: WESTERN BLOT
 They were then incubated with primary antibodies (anti-caspase
12, anti-Grp78 and anti-GAPDH, all at 1:1000 dilution) and
incubated in TBST and the corresponding primary antibody
(1:1000) overnight at 4°C
 The membranes were subsequently washed in TBST and
incubated for 1.5 hours at room temperature with the same
corresponding secondary antibody in a dilution of 1:3000 with
TBST
 Immunoreactive bands were visualized in the linear range with
enhanced chemoluminescence
 Films were scanned and the optical density was determined with
ImageJ

9. METHODS: ANALYSIS
 All statistical data shown was expressed as the mean optical
density for each treatment
 A one-way ANOVA test was used to compare means between
groups and determine statistical significance
 Differences of P<0.05 were considered statistically significant

10. RESULTS: CASPASE 12
 This figure shows the mean normalized optical density values
determined by ImageJ for Caspase 12
 GAPDH was used as the internal control to normalize values
Click to next slide for
more explanation
Legend
(50 mg protein lysate in each well)
1. Naïve- non-operated controls
2. Sham- operated controls not given focal ischemia
3. MCAO- rats were given focal ischemia by middle cerebral artery occlusion
4. MCAO+GCSF- rats were given focal ischemia by middle cerebral artery occlusion and administered GCSF

11. RESULTS: CASPASE 12
 The data demonstrates a relatively large decrease in expression
between the MCAO and MCAO+GCSF groups in the core and an
increase between groups in the penumbra
Caspase 12: 40 kDa
1 2 3 4
Legend
(50 mg protein lysate in each well)
1. Naïve- non-operated controls
2. Sham- operated controls not given focal ischemia
3. MCAO- rats were given focal ischemia by middle cerebral artery occlusion
4. MCAO+GCSF- rats were given focal ischemia by middle cerebral artery occlusion and administered GCSF

12. RESULTS: CASPASE 12
 ANOVA Tests were run for the results of both the core and
penumbra
 Since both had p values > .05, results were determined to be
statistically insignificant
Core
Penumbra

13. RESULTS: CASPASE 12
 A T-Test was then run which also proved results to be statistically
insignificant because the p value > .05

14. RESULTS: GRP78
 This figure displays the mean normalized optical density values
determined by ImageJ for GRP78
 GAPDH was used as the internal control to normalize values
Click to next slide for
more explanation
Legend
(50 mg protein lysate in each well)
1. Naïve- non-operated controls
2. Sham- operated controls not given focal ischemia
3. MCAO- rats were given focal ischemia by middle cerebral artery occlusion
4. MCAO+GCSF- rats were given focal ischemia by middle cerebral artery occlusion and administered GCSF

15. RESULTS: GRP78
 The data demonstrates a decrease in expression between the
MCAO and MCAO+GCSF groups in the core and an increase in
the penumbra
GRP78: 72-80 kDa
1 2 3 4
Legend
(50 mg protein lysate in each well)
1. Naïve- non-operated controls
2. Sham- operated controls not given focal ischemia
3. MCAO- rats were given focal ischemia by middle cerebral artery occlusion
4. MCAO+GCSF- rats were given focal ischemia by middle cerebral artery occlusion and administered GCSF

16. RESULTS: SUMMARY
 With both Caspase 12 and GRP78, there was a decrease in
expression in the treated rats in the core, but an increase in
expression in the treated rats in the penumbra

17. DISCUSSION: CASPASE 12
RESULTS
 In response to excess calcium in the ER lumen, an apoptotic
cascade is initiated involving Caspase 12 with the final death
marker prior to apoptosis being Caspase 3
 Consistent with previous studies, the core GCSF-treated rats
showed reduced Caspase 12 expression
 The penumbra GCSF-treated rats showed increased Caspase 12
expression in the penumbra in complete disagreement with
previous studies

18. DISCUSSION: GRP78 RESULTS
 In the absence of ER stress, the chaperone protein GRP78
protects cells from apoptosis by binding to IRE1, PERK, ATF6,
and Caspase 14
 GRP78 normally inhibits the activation of these intracellular
cytoplasmic kinases but when the ER is stressed, GRP78
dissociates from the cytoplasmic molecules to sequester the extra
calcium
 Similar to the results with Caspase 12, the core-GCSF treated
groups decreased GRP78 expression, but in the penumbra the
opposite effect occurred.

19. DISCUSSION: STATISTICS
 There was no statistical significance between any of the treatment
groups analyzed
 A possible reason for this result is the relatively small sample size of
data used in this experiment compared to similar studies
 Also there was a relatively high level of variation within each group,
which may have contributed to some of the observed discrepancies
in the data analysis
 Another statistical discrepancy may have come from GAPDH, which
was used to normalize OD values
 As demonstrated in Figure 1, the expression of GAPDH in the MCAO
group was significantly smaller than any other group making the
normalized value of the MCAO group relatively smaller

20. FUTURE RESEARCH
 In future research, additional Western Blots with Caspase 12 and
GRP78 would be run in order to compile more data and create
more reliable results
 As this research only investigated pro-death markers (Caspase
12) and ER stress indicators (GRP78), it would be interesting to
assay BCl2, a pro-survival marker in future research

21. SOURCES
 Http://www.weizmann.ac.il/immunology/Lapidot/documents/5.pdf (2002): n. pag. 17 June 2002.
Web.
 "Annals of Oncology." G-CSF Prevents the Suppression of Bone Marrow Hematopoiesis
Induced by IL-12 and Augments Its Antitumor Activity in a Melanoma Model in Mice. N.p., n.d.
Web. 26 Sept. 2012. <http://annonc.oxfordjournals.org/content/9/1/63.short>.
 "Phenotypic Changes in Neutrophil Granulocytes after G-CSF Administration in Patients with
Acute Lymphoblastic Leukemia under Chemotherapy." Phenotypic Changes in Neutrophil
Granulocytes after G-CSF Administration in Patients with Acute Lymphoblastic Leukemia
under Chemotherapy. N.p., n.d. Web. 26 Sept. 2012.
<http://www.haematologica.org/content/83/6/573.abstract>.
 "A Role for G-CSF ( Granulocyte-Colony Stimulating Factor ) Review ND ABBREVIATIONS
SC." Free Reference Manager and PDF Organizer. N.p., n.d. Web. 26 Sept. 2012.
<http://www.mendeley.com/research/a-role-for-gcsf-granulocytecolony-stimulating-factor-
review-nd-abbreviations-sc/>.
 Khan, Shaheen N., and Fridoon J. Ahmad. "IGF-1 and G-CSF Complement Each Other in
BMSC Migration towards Infarcted Myocardium in a Novel in Vitro Model." Cell Biology
International 33.6 (2009): n. pag. June 2009. Web. <IGF-1 and G-CSF complement each other
in BMSC migration towards infarcted myocardium in a novel in vitro model>.
 Chopp, M., S. Frinak, D. R. Walton, and M. B. Smith. "Intracellular Acidosis during and after
Cerebral Ischemia: In Vivo Nuclear Magnetic Resonance Study of Hyperglycemia in Cats."
N.p., n.d. Web. <http://stroke.ahajournals.org/content/18/5/919.full.pdf>.

22. SOURCES
 “Neuroprotective Effect of Recombinant Human Granulocyte Colony-stimulating Factor in Transient Focal Ischemia of
Mice." Nature.com. Nature Publishing Group, n.d. Web. 26 Sept. 2012.
 "Anti-apoptotic Effect of Granulocyte-colony Stimulating Factor after Focal Cerebral Ischemia in the Rat." Neuroscience
143.4 (2006): 965-74. 28 Dec. 2006. Web. <http://www.sciencedirect.com/science/article/pii/S0306452206012310>.
 Pan, Chunliu, Amit Gupta, Howard Prentice, and Jang-yen Wu. "Protection of Taurine and Granulocyte Colony-
Stimulating Factor against Excitotoxicity Induced by Glutamate in Primary Cortical Neurons." N.p., 2010. Web.
<http://www.biomedcentral.com/content/pdf/1423-0127-17-S1-S18.pdf>.
 "Neuroprotective Effect of Granulocyte Colonyâ Stimulating Factor After Focal Cerebral Ischemia." Neuroprotective
Effect of Granulocyte Colonyâ Stimulating Factor After Focal Cerebral Ischemia. N.p., n.d. Web. 26 Sept. 2012.
<http://stroke.ahajournals.org/content/34/3/745.long>.
 "Anti-apoptotic Effect of Granulocyte-colony Stimulating Factor after Focal Cerebral Ischemia in the Rat." Neuroscience
143.4 (2006): 965-74. 28 Dec. 2006. Web. <http://www.sciencedirect.com/science/article/pii/S0306452206012310>.
 "Caspase-12 Mediates Endoplasmic-reticulum-specific Apoptosis and Cytotoxicity by Amyloid-beta." National Center for
Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 26 Sept. 2012.
<http://www.ncbi.nlm.nih.gov/pubmed/10638761>.
 "Research." School of Biological and Biomedical Sciences Interorganellar Signalling Laboratory. N.p., n.d. Web.
<http://www.dur.ac.uk/martin.schroeder/Research%20Summary.htm>.
 "The Critical Roles of Endoplasmic Reticulum Chaperones and Unfolded Protein Response in Tumorigenesis and
Anticancer Therapies." Nature.com. Nature Publishing Group, n.d. Web. 26 Sept. 2012.
<http://www.nature.com/onc/journal/vaop/ncurrent/fig_tab/onc2012130f1.html>.