Granulocyte-colony stimulating factor in combination with stem cell factor confers greater neuroprotection after hypoxic-ischemic brain damage in the neonatal rats than a solitary treatment

Abstract

Neonatal hypoxia-ischemia (HI) is a devastating condition resulting in neuronal cell death and often culminates in neurological deficits. Granulocyte-colony stimulating factor (G-CSF) has been shown to have neuroprotective activity via inhibition of apoptosis and inflammation in various stroke models. Stem cell factor (SCF) regulates hematopoietic stem cells in the bone marrow and has been reported to have neuroprotective properties in an experimental ischemic stroke model. In this study we aim to determine the protective effects of G-CSF in combination with SCF treatment after experimental HI. Seven-day old Sprague-Dawley rats were subjected to unilateral carotid artery ligation followed by 2.5 hours of hypoxia. Animals were randomly assigned to five groups: Sham (n=8), Vehicle (n=8), HI with G-CSF treatment (n=9), HI with SCF treatment (n=9) and HI with G-CSF+SCF treatment (coadministration group; n=10). G-CSF (50 µg/kg), SCF (50 µg/kg) and G-CSF+SCF (50 µg/kg) were administered intraperitoneally 1 hour post HI followed by daily injection for 4 consecutive days (five total injections). Animals were euthanized 14 days after HI for neurological testing. Additionally assessment of brain, heart, liver, spleen and kidney atrophy was performed. Both G-CSF and G-CSF+SCF treatments improved body growth and decreased brain atrophy at 14 days post HI. No significant differences were found in the peripheral organ weights between groups. Finally, the G-CSF+SCF coadministration group showed significant improvement in neurological function. Our data suggest that administration of G-CSF in combination with SCF not only prevented brain atrophy but also significantly improved neurological function.

Keywords: Granulocyte-colony stimulating factor (G-CSF); Hypoxia-ischemia (HI); Neurological outcome; Stem cell factor (SCF).

Fig. 1. G-CSF and SCF improved body weight (a) and reduced brain atrophy (b) 14days post HI

Postnatal day-7 rats were subjected to HI. Intraperitoneal (IP) treatment with GCSF, SCF or G-CSF+SCF began at 1hour post HI and continued daily for 4days. (a) G-CSF, SCF and G-CSF+SCF treatment groups demonstrated an improved body weight (g) 14days post HI, with the combination treatment group showing best results. (b) Significant loss of right-to-left hemispheric (RH:LH) weight ratio is evident in vehicle rats and significantly improved with G-CSF and G-CSF+SCF treatment at 14days post HI (representative pictures shown; Data represent +/− SEM; *p<0.05 versus sham, #p<0.05 versus vehicle, †p<0.05 versus SCF ).

Fig. 2. Treatment groups showed no effect on organ weight at 14days post HI

Treatment did not appear to improve heart to body (a)liver to body (b)spleen to body (c) or kidney to body (d) weight ratios when measured 14days post HI. Although G-CSF+SCF treatment appeared to show some improvement in the above listed organs, statistical significance was not reached.

Fig. 3. G-CSF and SCF improved functional outcome at 14days post HI

The combination treatment group (G-CSF + SCF) showed significant improvement in neurological function according to (a) Modified Garcia test, where sensori-motor function was evaluated. Vehicle and SCF groups scored significantly worse on the 21 score scale system when compared to sham while the G-CSF+SCF treatment group showed significant improvement compared to vehicle and (b) Foot fault test, which shows the percentage of foot faults of the contralateral forelimb. The results show that ischemia-induced foot slips were significantly reduced by treatment with SCF and G-CSF+SCF (Data represent +/− SEM; *p<0.05 versus sham, #p<0.05 versus vehicle).