Effect of Human Umbilical Vein Endothelial Cells on Neurovascular Protection Against Hypoxic–Ischemia in Neonatal Rat Brain

• Main Authors: Yi-ChiChen, 陳奕淇
• Other Authors: Chao-Ching Huang
• Format: Others
• Language: en_US
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/16228990092252103224

碩士 === 國立成功大學 === 臨床醫學研究所 === 100 === Hypoxic-ischemia (HI) is a major cause of neonatal mortality and neurological morbidity among survivors. The neurovascular unit, composed of neurons, microvessels and microglia, is considered a major target of HI injury. Neurons and vascular endothelial cells may respond equally to the HI insult, and agents that simultaneously act on neuronal and endothelial cell protection may provide a powerful therapeutic strategy against HI. Human umbilical vein endothelial cells (HUVEC) may have the potential for treatment in the high-risk neonates for HI encephalopathy because of its regenerative potential and autologous capability. Stromal cell-derived factor 1 (SDF-1)/ C-X-C chemokine receptor type 4 (CXCR4) signaling and transmigration via blood-brain barrier are the key issue for the cell migration into the injured brain area. In our study, we hypothesized that peripheral injection of HUVEC entered the brain via SDF-1/CXCR4 pathway after HI, protected against neurovascular damage, and provided neuroprotection in neonatal brain. In vivo HI was induced by permanent ligation of unilateral carotid artery followed by 2-hours of hypoxia in postpartum day 7 Sprague-Dawley rat pups. Rat pups received intraperitoneal injections of HUVECs (1×105/per injection), conditioned medium or saline solution before and immediately after HI. The littermates were divided into five groups: control group, HUVEC-P4-treated group (Low passage), HUVEC-P8-treated group (High passage), condition medium-treated group, and normal saline-treated group. In vitro oxygen-glucose deprivation (OGD) was established on mouse neuroblastoma neuronal cells (Neuro-2a) and mouse immortalized cerebral vascular endothelial cells (b.End3). The Neuro-2a cells and b.End3 cells were then co-cultured, respectively, with HUVEC-P4 before OGD. We found that intraperitoneally-injected HUVEC-P4 entered the ipsilateral cerebral cortex after HI and positioned closed to the neurons and microvessels. Compared with the condition medium-treated group, the HUVEC-P4 but not the HUVEC-P8 group showed significantly less neuronal loss and more preservation of microvessels in the cortex 24 hours after HI. BBB damage, determined by IgG extravasation, was also significantly reduced in the HUVEC-P4 but not the HUVEC-P8 group. Compared with HUVEC-P8, HUVEC-P4 had higher migratory properties, in addition, the HUVEC-P4-treated group had more GFP-positive cells in the circulation at 3 hours after injection than the HUVEC-P8-treated group. Seven days after injury, the HUVEC-P4 but not the HUVEC-P8 group had significantly decreased brain volume loss in the cortex and striatum compared with the condition medium or saline-treated group. SDF-1 was up-regulated in the ipsilateral cortex three hours after HI, and inhibiting the SDF-1/CXCR4 axis reduced the neuroprotective effect provided by HUVEC-P4. Co-culturing of HUVEC-P4 protected against OGD cell death in both neuronal cells and vascular endothelial cells. In conclusion, our study indicates that peripherally-injected HUVEC-P4 enters the neonatal brain after HI via SDF-1/CXCR4 pathway, protects against neurovascular damage, and provides long-term neuroprotection. Cell therapy using HUVECs may provide a powerful therapeutic strategy in the treatment of high-risk neonates with HI injury. Elucidating the shared neuronal and vascular protective mechanism mediated by HUVEC may yield neurovascular protective drugs that mimic the beneficial effects of HUVEC for treating high-risk newborns with asphyxia.