THE EFFECTS OF

GRANULOCYTE

COLONY

STIMULATING

FACTOR ON

STROKE INDUCED

RATSMatthew Busel

Pine Crest School

Work conducted at Florida Atlantic University

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: 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

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 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 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

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 insignificantCore

Penumbra

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

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

Click to next slide for

more explanation

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

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 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

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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