THE EFFECTS OFGRANULOCYTECOLONYSTIMULATINGFACTOR ONSTROKE INDUCEDRATSMatthew BuselPine Crest SchoolWork conducted at Florida AtlanticUniversity
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
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
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
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
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
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)
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
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
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
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
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
RESULTS: CASPASE 12 A T-Test was then run which also proved results to be statistically insignificant because the p value > .05
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
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
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
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
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.
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
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
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