Yonsei Med J.  2008 Oct;49(5):836-842. 10.3349/ymj.2008.49.5.836.

Granulocyte Stimulating Factor Attenuates Hypoxic-ischemic Brain Injury by Inhibiting Apoptosis in Neonatal Rats

• Kim BR
• Shim JW
• Sung DK
• Kim SS
• Jeon GW
• Kim MJ
• Chang YS
• Park WS
• Choi ES

• KMID: 2158203
• DOI: http://doi.org/10.3349/ymj.2008.49.5.836

Abstract

PURPOSE
This study was undertaken to determine the neuroprotective effect of granulocyte stimulating factor (G-CSF) on neonatal hypoxic-ischemic brain injury. MATERIALS AND METHODS: Seven-day-old male newborn rat pups were subjected to 110 minutes of 8% oxygen following a unilateral carotid artery ligation. Apoptosis was identified by performing terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and flow cytometry with a combination of fluorescinated annexin V and propidium iodide (PI) and JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide). The extent of cerebral infarction was evaluated at 2 weeks after recovery. RESULTS: With a single dose (50microgram/kg) of G-CSF treatment immediately after hypoxic-ischemic insult, hypoxia-ischemia induced increase in TUNEL-positive cells, annexinV+/PI- and JC-1 positive apoptotic cells in the ipsilateral cerebral cortex was significantly reduced at 24 hours, measured by flow cytometry, and the extent of cerebral infarction at 2 weeks after recovery was also significantly attenuated compared to the hypoxia-ischemia control group. CONCLUSION: Our data suggest that G-CSF is neuroprotective by inhibiting apoptosis, thereby reducing the ensuing cerebral infarction in a newborn rat pup model of cerebral hypoxia-ischemia (HI).

Keyword

Granulocyte stimulating factor; neuroprotective agents; hypoxia-ischemia; newborn; apoptosis

MeSH Terms

Animals
Apoptosis/*drug effects
Brain/pathology
Cerebral Infarction/pathology/prevention & control
Flow Cytometry
Granulocyte Colony-Stimulating Factor/*pharmacology/therapeutic use
Hypoxia-Ischemia, Brain/*drug therapy/pathology
In Situ Nick-End Labeling
Male
Organ Size
Protective Agents/*pharmacology/therapeutic use
Rats
Rats, Sprague-Dawley
Weight Gain

Figure

• 

  Fig. 1 Representative photomicrographs of TUNEL staining (A) at the anterior portion of the ipsilateral cerebral cortex (× 400) and number of TUNEL-positive cells (B) per high power field in the NC, HC, and HG groups at 24 hours after HI. Note significantly increased TUNEL-positive green cells in HC compared to NC and less TUNEL-positive cells in HG compared to HC. All data are mean ± standard deviation. *p < 0.05 compared to NC. †p < 0.05 compared to HC. NC, normoxia control group; HC, HI control group; HG, HI with G-CSF treatment group.

• 

  Fig. 2 (A) Representative flow cytogram of an annexin V binding (abscissa) versus PI uptake (ordinate) in the ipsilateral cerebral hemisphere of newborn rat brain cells at 24 hours after HI. The numbers in the upper left quadrant (Q1), upper right quadrant (Q2), lower left quadrant (Q3), and lower right quadrant (Q4) represent the percentage of damaged (annexin V-/PI+), necrotic (annexin V+/PI+), live (annexin V-/PI-) and apoptotic cells (annexin V+/PI-), respectively. (B) Representative flow cytometric analyses of mitochondrial membrane potential using JC-1. The shift of JC-1 fluorescence from orange (FL2) to green (FL1) indicates a collapse of mitochondrial membrane potential. NC, normoxia control group; HC, HI control group; HG, HI with G-CSF treatment group.

• 

  Fig. 3 Body weight gain, indicative of general well being, in the rat pups of each experimental group. Significantly less body weight gain was observed in HC compared to NC staring from 3 days after HI, and was improved in HG from 10 days after HI compared to HC. NC, normoxia control group; HC, HI control group; HG, HI with G-CSF treatment group. All data are mean ± standard deviation. *p < 0.05 compared to NC. †p < 0.05 compared to HC.

• 

  Fig. 4 Representative photomicrographs of hematoxylin/eosin staining (A) at the anterior commisure and percentage of the preserved brain volume (B) from the normoxia control (NC), HI control (HC), and HI with G-CSF treatment (HG) groups at 2 weeks after the injury. Significantly increased infarct volume in HC compared to NC, and significantly reduced infarct volume of HG compared to HC are observed. NC, normoxia control group; HC, HI control group; HG, HI with G-CSF treatment group. All values are mean ± standard deviation. *p < 0.05 compared to NC. †p < 0.05 compared to HC.

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