1. THE EFFECTS OFGRANULOCYTECOLONYSTIMULATINGFACTOR ONSTROKE INDUCEDRATSMatthew BuselPine Crest SchoolWork conducted at Florida AtlanticUniversity

2. INTRODUCTION: GCSF Granulocyte Colony Stimulating Factor (GCSF) is a growth factorand cytokine that stimulates the proliferation and mobilization ofhematopoietic 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 thebrain leading to neuroprotective effects of the drug By decreasing the rate of neuronal death many central nervoussystem (CNS) disorders may be counteracted against 3. INTRODUCTION: CEREBRAL ISCHEMIA Focal cerebral ischemia (stroke) is a CNS disorder in which thecerebral blood vessels are blocked, resulting in an inadequateblood flow to the brain and subsequent oxygen (hypoxic) glucose deprived environment In this state the brain cannot meet metabolic demands andneuronal death will eventually be initiated via apoptosis The resulting ischemic infarct (area of dead cell tissue) includes acentral core (an area of severe ischemia) and a surroundingpenumbra, which is vulnerable if the cell death is not arrested White = core Red = penumbra 4. INTRODUCTION: APOPTOTICPATHWAYS The endoplasmic reticulum (ER) is responsible for the proper foldingand sorting of proteins During an ischemic insult the ER becomes stressed and will try torestore normal function by deactivating protein translation signalingpathways while activating other pathways that increase theproduction of chaperone molecules that facilitates protein folding If it is unsuccessful in rectifying its stressed state and restoringnormal function, the ER will initiate an apoptotic cascade Caspase 12 is an ER-membrane associated protein and one of theinitiators of the caspase-mediated apoptotic pathway Glucose regulated protein 78 (GRP78) is a chaperon moleculeassociated with the ER 5. INTRODUCTION: EXPERIMENT The molecular expression of GRP78 and Caspase 12 werecompared in GCSF treated and untreated rats Both GRP78 and Caspase 12 are excellent indicators of celldeath 6. HYPOTHESIS If the molecular expression of GRP78 and Caspase 12 aremeasured in GCSF treated and untreated rats after focal cerebralischemia, treated rats will have lower expression of the selectedindicators and therefore lower cell death in both the core andpenumbra of the brain 7. METHODS: WESTERN BLOT Frozen homogenates/lysates of protein samples (from two rats toincrease diversity with sample population) were used to performwestern blot assays Briefly, protein homogenates/lysates were boiled for 5 minutes andcentrifuged Each lane was loaded with 50 g of protein and subjected to sodiumdodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) on12% gels and then electrophoretically transferred to nitrocellulosemembranes Once protein transfers were completed, the nitrocellulosemembranes were blocked in 5% nonfat dry milk in TBST (20 mmol/LTris base, pH 7.6, 137 mmol/L NaCl, and 0.05% Tween 20) for 1 hourat 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-caspase12, anti-Grp78 and anti-GAPDH, all at 1:1000 dilution) andincubated in TBST and the corresponding primary antibody(1:1000) overnight at 4C The membranes were subsequently washed in TBST andincubated for 1.5 hours at room temperature with the samecorresponding secondary antibody in a dilution of 1:3000 withTBST Immunoreactive bands were visualized in the linear range withenhanced chemoluminescence Films were scanned and the optical density was determined withImageJ 9. METHODS: ANALYSIS All statistical data shown was expressed as the mean opticaldensity for each treatment A one-way ANOVA test was used to compare means betweengroups and determine statistical significance Differences of P .05, results were determined to bestatistically insignificantCorePenumbra 13. RESULTS: CASPASE 12 A T-Test was then run which also proved results to be statisticallyinsignificant because the p value > .05 14. RESULTS: GRP78 This figure displays the mean normalized optical density valuesdetermined by ImageJ for GRP78 GAPDH was used as the internal control to normalize valuesClick to next slide formore explanationLegend(50 mg protein lysate in each well)1.Nave- non-operated controls2.Sham- operated controls not given focal ischemia3.MCAO- rats were given focal ischemia by middle cerebral artery occlusion4.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 penumbraGRP78: 72-80 kDa1 2 3 4Legend(50 mg protein lysate in each well)1. Nave- non-operated controls2. Sham- operated controls not given focal ischemia3. MCAO- rats were given focal ischemia by middle cerebral artery occlusion4. 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 inexpression in the treated rats in the core, but an increase inexpression in the treated rats in the penumbra 17. DISCUSSION: CASPASE 12RESULTS In response to excess calcium in the ER lumen, an apoptoticcascade is initiated involving Caspase 12 with the final deathmarker prior to apoptosis being Caspase 3 Consistent with previous studies, the core GCSF-treated ratsshowed reduced Caspase 12 expression The penumbra GCSF-treated rats showed increased Caspase 12expression in the penumbra in complete disagreement withprevious studies 18. DISCUSSION: GRP78 RESULTS In the absence of ER stress, the chaperone protein GRP78protects cells from apoptosis by binding to IRE1, PERK, ATF6,and Caspase 14 GRP78 normally inhibits the activation of these intracellularcytoplasmic kinases but when the ER is stressed, GRP78dissociates from the cytoplasmic molecules to sequester the extracalcium Similar to the results with Caspase 12, the core-GCSF treatedgroups decreased GRP78 expression, but in the penumbra theopposite effect occurred. 19. DISCUSSION: STATISTICS There was no statistical significance between any of the treatmentgroups analyzed A possible reason for this result is the relatively small sample size ofdata 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 discrepanciesin the data analysis Another statistical discrepancy may have come from GAPDH, whichwas used to normalize OD values As demonstrated in Figure 1, the expression of GAPDH in the MCAOgroup was significantly smaller than any other group making thenormalized value of the MCAO group relatively smaller 20. FUTURE RESEARCH In future research, additional Western Blots with Caspase 12 andGRP78 would be run in order to compile more data and createmore reliable results As this research only investigated pro-death markers (Caspase12) and ER stress indicators (GRP78), it would be interesting toassay 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 HematopoiesisInduced by IL-12 and Augments Its Antitumor Activity in a Melanoma Model in Mice. N.p., n.d.Web. 26 Sept. 2012. . "Phenotypic Changes in Neutrophil Granulocytes after G-CSF Administration in Patients withAcute Lymphoblastic Leukemia under Chemotherapy." Phenotypic Changes in NeutrophilGranulocytes after G-CSF Administration in Patients with Acute Lymphoblastic Leukemiaunder Chemotherapy. N.p., n.d. Web. 26 Sept. 2012.. 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