Protective Mechanisms of Granulocyte-Colony Stimulating Factor Against Experimental Models of Stroke

Ischemic stroke has a multiplicity of pathophysiological mechanisms.
Granulocyte-colony stimulating factor (G-CSF) is an endogenous growth factor that
exerts a diverse range of neuroprotection against ischemic stroke. Several lines of
evidence demonstrated the contribution of endoplasmic reticulum (ER) in apoptotic cell
death involving ischemia. Cell culture of undifferentiated PC12 cells were subjected to
10mM glutamate and selected doses of G-CSF (25ng/ml, 50ng/ml, 100ng/ml and
250ng/ml) for 24 hours. Cell viability, expression of the G-CSF receptor and expression
level of CHOP were assessed in vitro. Sprague-Dawley rats were subjected to middle
cerebral artery occlusion (MCAO). Rats were subcutaneously injected with G-CSF (n=
15; 50ug/kg body weight) 24 hours post-MCAO for 4 days. Vehicle treated rats were
administered 5% dextrose for 1 day (n=4) or 4 days (n=16). Sham-operated rats (n=9)
were not subjected to MCAO. Neurological deficit and infarct volume were measured while expression levels of pAKT, Bcl2, Bax, Bak, cleaved caspase-3, GRP78, ATF4,
ATF6, p-p38MAPK, pJNK, CHOP and HSP27 were analyzed by western blotting. In
vitro G-CSF receptor was expressed on undifferentiated PC12 cell, and an optimal dose
of 50 ng/ml G-CSF significantly protected these cells against glutamate-induced
cytotoxicity (P < 0.05). G-CSF significantly down-regulated (P < 0.01) the ER stressinduced
pro-apoptotic marker CHOP in vitro. In vivo, G-CSF reduced infarct volume to
50% while significantly improved neurological deficit compared to vehicle rats. G-CSF
significantly (P < 0.05) up-regulated pro-survival proteins pAKT and Bcl2 while downregulating
pro-apoptotic proteins Bax, Bak and cleaved caspase 3 in the ischemic brain.
It also significantly (P < 0.05) downregulated the ER intraluminal stress sensor GRP78,
proteins of ER stress induced intracellular pathway; ATF4, ATF6, p-p38MAPK, pJNK
and the ER stress induced apoptotic marker CHOP, which suggests that ER stress is
being ameliorated by G-CSF treatment. G-CSF also reduced the level of HSP27,
providing additional evidence of cellular stress reduction. G-CSF treatment increased
cell survival by attenuating both general pro-apoptotic proteins and specific effector
proteins in the ER stress induced apoptotic pathways. Our data has provided new insight
into the anti-apoptotic mechanism of G-CSF, especially as it relates to ER stress induced
apoptosis in ischemia.