THE EFFECTS OFGRANULOCYTECOLONYSTIMULATINGFACTOR ONSTROKE INDUCEDRATSMatthew BuselPine Crest SchoolWork conducted at Flori...
INTRODUCTION: GCSF Granulocyte Colony Stimulating Factor (GCSF) is a growth factor  and cytokine that stimulates the prol...
INTRODUCTION: CEREBRAL ISCHEMIA Focal cerebral ischemia (stroke) is a CNS disorder in which the  cerebral blood vessels a...
INTRODUCTION: APOPTOTICPATHWAYS The endoplasmic reticulum (ER) is responsible for the proper folding    and sorting of pr...
INTRODUCTION: EXPERIMENT The molecular expression of GRP78 and Caspase 12 were  compared in GCSF treated and untreated ra...
HYPOTHESIS If the molecular expression of GRP78 and Caspase 12 are  measured in GCSF treated and untreated rats after foc...
METHODS: WESTERN BLOT Frozen homogenates/lysates of protein samples (from two rats to  increase diversity with sample pop...
METHODS: WESTERN BLOT They were then incubated with primary antibodies (anti-caspase  12, anti-Grp78 and anti-GAPDH, all ...
METHODS: ANALYSIS All statistical data shown was expressed as the mean optical  density for each treatment A one-way ANO...
RESULTS: CASPASE 12      This figure shows the mean normalized optical density values        determined by ImageJ for Cas...
RESULTS: CASPASE 12   The data demonstrates a relatively large decrease in expression      between the MCAO and MCAO+GCSF...
RESULTS: CASPASE 12  ANOVA Tests were run for the results of both the core and    penumbra  Since both had p values > .0...
RESULTS: CASPASE 12 A T-Test was then run which also proved results to be statistically  insignificant because the p valu...
RESULTS: GRP78      This figure displays the mean normalized optical density values        determined by ImageJ for GRP78...
RESULTS: GRP78    The data demonstrates a decrease in expression between the       MCAO and MCAO+GCSF groups in the core ...
RESULTS: SUMMARY With both Caspase 12 and GRP78, there was a decrease in  expression in the treated rats in the core, but...
DISCUSSION: CASPASE 12RESULTS In response to excess calcium in the ER lumen, an apoptotic  cascade is initiated involving...
DISCUSSION: GRP78 RESULTS In the absence of ER stress, the chaperone protein GRP78  protects cells from apoptosis by bind...
DISCUSSION: STATISTICS There was no statistical significance between any of the treatment    groups analyzed   A possibl...
FUTURE RESEARCH In future research, additional Western Blots with Caspase 12 and  GRP78 would be run in order to compile ...
SOURCES Http://www.weizmann.ac.il/immunology/Lapidot/documents/5.pdf (2002): n. pag. 17 June 2002.    Web.   "Annals of ...
SOURCES   “Neuroprotective Effect of Recombinant Human Granulocyte Colony-stimulating Factor in Transient Focal Ischemia ...
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Sigma xi presentation

  1. 1. THE EFFECTS OFGRANULOCYTECOLONYSTIMULATINGFACTOR ONSTROKE INDUCEDRATSMatthew BuselPine Crest SchoolWork conducted at Florida AtlanticUniversity
  2. 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. 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. 4. INTRODUCTION: APOPTOTICPATHWAYS 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. 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. 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. 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. 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. 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. 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 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
  11. 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 penumbraCaspase 12: 40 kDa1 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. 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 insignificantCorePenumbra
  13. 13. RESULTS: CASPASE 12 A T-Test was then run which also proved results to be statistically insignificant because the p value > .05
  14. 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 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. 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 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. 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. 17. DISCUSSION: CASPASE 12RESULTS 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. 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. 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. 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. 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. 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>.

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