The Genes Of COX-2,GluR2 And PAFR Expression Of The Primary Cultured Hippocampal Neurons Of Rats Following Fluid Percussion Injury In Vitro

Objective: To study the expressions of platelet activating factor receptor(PAFR), cyclooxygenase-2(COX-2) and glutamate receptor 2 (GluR2) in the primary cultured hippocampal neurons of rats following fluid percussion injury in vitro,and to observe the temporal patterns and relationship among their expressions and localizations following injury.Methods: Hippocampus neurons of Sprague-Dawley neonatal rats (24h) were cultured in vitro, and then were identified by double immunofluorescence staining with anti-MAP2 and anti-GFAP. The cultured hippocampus neurons with growth in good condition were randomly divided into control group and injury groups which were subjected to fluid percussion injury and then subdivided into 4h,8h,12h,24h and 48h groups according to the time elapsed after injury. The morphological changes of hippocampus neurons after fluid percussion injury were observed with transmission electron microscope (TEM). The expressions of PAFR,COX-2 and GluR2 on mRNA level were studied by reverse transcription polymerase chain reaction (RT-PCR). The localization of PAFR,COX-2 and GluR2 in cultured hippocampus neurons were studied with double immunofluorescence staining .Results: 1 The Morphological findings: After cultured 7 days, mature hippocampal neurons showed abundant cytoplasm, good refraction, nuclei were often located in the center of the cell or leaned to one side, nucleus and nucleolus were clear, neurite and dendrite of neurons interlaced into reticulate.2 Identification of neurons: Double immunofluorescence staining with anti-MAP2 and anti-GFAP demonstrated that 70%-80% of the cultured cells were neurons.3 The results of H.E Staining: Following the fluid percussion injury (0.2MPa), the population of neurons obviously decreased. The morphology of injured cells appeared significant change, such as neurite and dendrite obviously shortern, cytoplasm of deep red colour, nucleus condensed.4 The ultrastructure changes: Following the fluid percussion injury (0.2MPa), ultrastructure of injured neurons showed swelling cytoplasm, cell organelle obviously decreased, many inequality of size and round or ovi-round vacuoles in cytoplasm, lack cristae or no cristae of most mitochondria. Rough endoplasmic reticulum dilatated as lakes and degranulated. The decrease of polyribosomes and nuclear membrane disorganization were observed. The ultrastructure changes of injured neurons were most serious at 12h after injury.5 The results of RT-PCR:(1) COX-2: At 4h after injury, the expression of COX-2 increased, which was highest at 12h, and remained higher than control group (p<0.05).(2) GluR2: At 4h after injury, the expression of GluR2 decreased, which was lowest at 8h, and remained lower than control group (p<0.05).(3) PAFR: At 4h after injury, the expression of PAFR decreased, which was lowest at 8h, and remained lower than control group (p<0.05).6 The results of immunofluorescence staining:(1) The immunofluorescence staining of COX-2: MAP2 marked hippocampus neurons showed red fluorescence. COX-2 positive cells showed green fluorescence. COX-2 was located in cell membrane, cytoplasm, neurite and dendrite of hippocampus neurons.(2) The immunofluorescence staining of GluR2: MAP2 marked hippocampus neurons showed red fluorescence. GluR2 positive cells showed green fluorescence. GluR2 was mainly located in neurite, dendrite and the origin of neurite of cytoplasm hippocampus neurons.(3) The immunofluorescence staining of PAFR: MAP2 marked hippocampus neurons showed red fluorescence. PAFR positive cells showed green fluorescence. PAFR was mainly located in cell membrane, neurite and dendrite of hippocampus neurons.Conclusion: 1 After fluid percussion injury for 4h, the expression of COX-2 in cultured hippocampus neurons increased progressively, and reached the peak level after 12h. It was more than the normal after hippocampus neurons injury for 48h. COX-2 was located in cell membrane, cytoplasm, neurite and dendrite of hippocampus neurons. 2. After fluid percussion injury for 4h, the expression of GluR2 in cultured hippocampus neurons decreased progressively, and reached the lowest level after 8h. It was less than the normal until hippocampus neurons injury for 48h. GluR2 was mainly located in neurite, dendrite and the origin of neurite of cytoplasm hippocampus neurons. 3. After fluid percussion injury for 4h, the expression of PAFR in cultured hippocampus neurons decreased progressively, and then reached the lowest level after 8h. It was less than the normal until hippocampus neurons injury for 48h. PAFR was mainly located in cell membrane, neurite and dendrite of hippocampus neurons. 4.The similar temporal patterns and localization of COX-2,GluR2 and PAFR expressions following injury were observed in cultured hippocampus neurons. It is suggested that there was a close relationship among COX-2,GluR2 and PAFR during fluid percussion injury. The expressions of COX-2,GluR2 and PAFR had important roles in the progression of hippocampus neurons injury. The study of expressions of COX-2,GluR2 and PAFR would be helpful for the treatment of early mechanical brain injury.