Effects Of Granulocyte Colony Stimulating Factor Mobilize Endothelial Progenitor Cells On Functional Recovery After Traumatic Brain Injury In A Rat Model

Objective:Endothelial progenitor cells (EPCs) maintain the physiologic functions of normal vascular system; they also play a pivotal role in vasculogenesis and angiogenesis in pathologic condition. However, there is rare study of EPCs related angiogenesis in traumatic brain injury (TBI). Granulocyte colony-stimulating factor (G-CSF) is well-established to mobilize EPCs which proliferate and differentiat precursor cells of endotheliocyte from bone marrow into the peripheral blood, participates repair and regeneration of injure tissues. G-CSF becomes popular in nervous system disease. To investigate the effect of G-CSF on the neurofunctional protection, we hope to find a new way to treat craniocerebral trauma.Material and Methods:1.36 of 300-350g and 7 week age adult male Wistar rats (from Academy of Military Medical of China) were randomly divided into three groups:Sham group (12), normal saline group (NS,12) and rhG-CSF group (12). rats were anesthetized with 10% chloral hydrate (0.3ml/kg) administered intraperitoneally and then placed in a stereotaxic frame. A 4.0 mm craniotomy was performed over the right parietal skull to expose the dura (4.4 mm posterior from bregma and 2.4 mm lateral to the sagittal suture). The pressure pulse of the fluid percussion device between 2.0 to 2.5 atm. Sham group grinds the skull but not to percuss. RhG-CSF group was administered G-CSF (50μg/kg; Chugai Pharmaceutical, Co., Japan) subcutaneously for 5 consecutive days after TBI. NS group mitte tales doses normal saline. The 0.5ml blood samples was collected from the rat eyes at 6 hr, 1day,3 day and 6 day after TBI. Blood samples were first subjected to a Ficoll gradient centrifugation to isolate mononuclear cells using a commercial kit. The isolated cells suspended in BSA buffer were incubated with a both PE-conjugated CD34 antibody and FITC-conjugated CD133 antibody for 10 minutes at room temperature. Then the staining cells were analyzed in a flow cytometry. The modified neurological severity score tests were carried out on preinjury and on days 1,4,7,14, and 21 after TBI. The Morris water maze test was tested on days 21-25 after TBI.2.56 of adult male Wistar rats were randomly divided into NS group and rhG-CSF group,28 rats in each group. CD31-MVD were measured by immunohistochemistry in boundary zone of lesion and the CA3 region of the hippocampus on days 4,7,14 and 21 after TBI. Random 7 rats were divided in each time piont.Results:1. There were double CD34/CD133 positive cells, which were defined as EPCs, in the circulating blood in rats. The numbers of circulating EPCs increased significantly at 6 hr after TBI, recurred to normal level after 1 day. After G-CSF mobilization, the numbers of circulating EPCs increased significantly and gradully increased with injection frequency.2. Injury in the hemisphere cortex of rats causes neurological functional deficits as measured by mNSS. The mNSS scores for the rhG-CSF group were significantly decreased at days 14 and 21 after TBI when compared with the NS group.3. The dysfunction of spatial learning were found by the Morris water maze test. the rhG-CSF group reduced the dysfunction of spatial learning caused by the brain damage in this model. The percentage of time spent in the correct quadrant was significantly higher and escape latency was significantly lower in the rhG-CSF group than the NS group during the water maze test.4. CD31-MVD was measured in boundary zone of lesion and the CA3 region of the hippocampus. The number of CD31-MVD was highest at 7 day in the NS group, significantly higher in the rhG-CSF group than the NS group at 7, 14, and 21 days after TBI.Conclusion:1. EPCs can be mobilized to the circulating blood from bone marrow after TBI. The numbers of circulating EPCs were increased significantly by G-CSF after TBI.2. Injury in the hemisphere cortex of rats causes neurological functional deficits and spatial learning disfunction. These disfunctions were recoveried by G-CSF.3. Circulating EPCs were mobilized and homing by G-CSF. The higher EPCs level conduces to augment more angiogenesis and functional recovery in the injured brain after TBI.