Effect of granulocyte-colony stimulating factor on endoplasmic reticulum stress in neonatal rats after hypoxic-ischemic brain damage

Abstract

Objective To evaluate the protective effect of granulocyte-colony stimulating factor(G-CSF) on neonatal rats after hypoxic-ischemic brain damage(HIBD)and its effect on endoplasmic reticulum (ER) stress. Methods According to the random number table, a total of 54 Sprague-Dawley (SD) rats aged 7 days were divided into 3 groups(18 rats in each group): Sham group, HIBD group and G-CSF group, and the improved Rice method was used to establish a neonatal rat model of HIBD.A dose of 50 μg/kg of G-CSF was administered intraperitoneally 1 hour after HIBD (G-CSF group), while the rats in HIBD group and Sham group received saline only.At 24 hours of HIBD, pups were euthanized to quantify brain infarct volume by using 2, 3, 5-Triphenyltetrazolium chloride.Hematoxylin-Eosin (HE) staining was used to observe the changes of brain structure.Neuronal cell death was determined by using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Then the expressions of glucose-regulated protein 78 (GRP78), cysteinyl aspartate specific proteinase 12 (Caspase-12), CCAAT/enhancer binding-protein homologous protein (CHOP) were assessed by Western blot and immunofluorescence staining. Results Twenty-four hours after operation, HE staining showed that no significant neuronal damage was observed in Sham group.The brain tissue structure of rats in the HIBD group was significantly damaged, while some improvement was observed in the G-CSF group.The infarction volume in HIBD group[(25.40±5.15)%] increased compared with that in the Sham group[(0.31±0.15)%] and the G-CSF group[(16.36±4.97)%], and the differences were statistically significant(all P<0.05). There was increased positive expression of GRP78 protein in HIBD group, compared with that in the Sham group and the G-CSF group[(49.38±10.06)% vs.(9.12±4.50)%, (30.61±6.35)%], and the differences were statistically significant (all P<0.05). The percentage of apoptosis in the hippocampal CA1 region and conex in HIBD group [(44.84±11.54)%, (48.23±14.07)%] were higher than those in the G-CSF group [(17.87±7.20)%, (26.18±9.96)%], and the differences were statistically significant (all P<0.05). The expression of GRP78, CHOP and Caspase-12 in the HIBD group (0.63±0.24, 0.72±0.21, 0.68±0.25) were higher than those in the Sham group (0.20±0.08, 0.28±0.08, 0.23±0.07), and the G-CSF group (0.39±0.13, 0.51±0.18, 0.48±0.16), and the differences were statistically significant (all P<0.05). Conclusions G-CSF exerts neuroprotective effect on the neonatal rats after HIBD.G-CSF may be an effective treatment of HIBD by reducing ER stress-induced neuronal apoptosis. Key words: Infant, newborn; Hypoxic-ischemic brain damage; Granulocyte-colony stimulating factor; Endoplasmic reticulum stress