Intranasal administration of Cytoglobin modifies human umbilical cord-derived mesenchymal stem cells and improves hypoxic-ischemia brain damage in neonatal rats by modulating p38 MAPK signaling-mediated apoptosis

Neonatal hypoxic-ischemic brain damage (HIBD) is a common clinical syndrome in newborns. Hypothermia is the only approved therapy for the clinical treatment; however, the therapeutic window of hypothermia is confined to 6 h after birth and even then, >40% of the infants either die or survive with various impairments, including cerebral palsy, seizure disorder and intellectual disability following hypothermic treatment. The aim of the present study was to determine whether nasal transplantation of Cytoglobin (CYGB) genetically modified human umbilical cord-derived mesenchymal stem cells (CYGB-HuMSCs) exhibited protective effects in neonatal rats with HIBD compared with those treated without genetically modified CYGB. A total of 120 neonatal Sprague-Dawley rats (postnatal day 7) were assigned to either a Sham, HIBD, HuMSCs or CYGB-HuMSCs group (n = 30 rats/group). For HIBD modeling, rats underwent left carotid artery ligation and were exposed to 8% oxygen for 2.5 h. A total of 30 min after HI, HuMSCs (or CYGB-HuMSCs) labeled with enhanced-green fluorescent protein (eGFP) were intranasally administered. After modeling for 3, 14 and 29 days, five randomly selected rats were sacrificed in each group, and the expression levels of CYGB, ERK, JNK and p38 in brain tissues were determined. Nissl staining of the cortex and hippocampal Cornu Ammonis 1 area of rats in each group were compared after 3 days of modeling. TUNEL assay and immunofluorescence were performed 3 days after modeling. Long term memory in rats was assessed using a Morris-water maze 29 days after modeling. The HIBD group demonstrated significant deficiencies compared with the Sham group based on Nissl staining, TUNEL assay and the Morris-water maze test. HuMSC treated rats exhibited improvement on in all the tests, and CYGB-HuMSCs treatment resulted in further improvements. PCR and western blotting results indicated that the CYGB mRNA and protein levels were increased from day 3 to day 29 after transplantation of CYGB-HuMSCs. Furthermore, it was identified that CYGB-HuMSC transplantation suppressed p38 signaling at all experimental time points. Immunofluorescence indicated the scattered presence of HuMSCs or CYGB-HuMSCs in damaged brain tissue. No eGFP and glial fibrillary acidic protein or eGFP and neuron-specific enolase double-stained positive cells were found in the brain tissues. Therefore, CYGB-HuMSCs may serve as a gene transporter, as well as exert a neuroprotective and antiapoptotic effect in HIBD, potentially via the p38 mitogen-activated protein kinase signaling pathway.