Effects Of Epidermal Growth Factor Receptor Inhibitors On Microenvironment Of Axonal Regeneration After Spinal Cord Injury In Rats

Part one The effects of epidermal growth factor receptor inhibitor AG1478 on GAP-43 expression and motor functional impairment after spinal cord injury in ratsObjective The formation of glial scar and production of myelin-associated inhibitory molecules are two main obstacles to axonal regeneration after spinal cord injury. Recent studies have indicated that epidermal growth factor receptor (EGFR) activation triggered quiescent astrocytes into becoming reactive astrocytes and forming glial scar. And epidermal growth factor receptor inhibitors AG1478 promoted the axonal regeneration post optic injury by attenuating the inhibition effects of myelin-associated inhibitor molecule and chondroitin sulfate proteoglycans of axon. Therefore epidermal growth factor receptor inhibitors may attenuate the function impairment by promoting the injury axon regeneration after spinal cord injury. In these experiments we would administer the epidermal growth factor receptor inhibitors AG1478 in the local lesion and then observed whether AG1478 could upregulate the expression of growth associated protein-43(GAP-43) and attenuate the function impairment post injury.Methods The rats spinal cord injury model were built by using the weight-drop method, and sham-operated animals underwent T10 laminectomy alone.48 SD health female adult rats were randomly divided into 3 equal groups:sham group, injury control group and AG1478-treated group. The rats in AG1478-treated group were locally administered 0.2 ml 2mM AG1478 using a Gelfoam as a carrier, while the sham group and control group were locally administered dimethylsulfoxid also using a Gelfoam as a carrier. Immunofluorescence staining and Western blotting was used to detect the expression of growth associated protein-43 (GAP-43) 28 day post injury. The BBB scores of the hindlimb locomotor were collected at the first day post injury and weekly afterward (8 weeks). The body weight of all rats was measured previous injury and weekly afterward post injury (8 weeks).Results Immunofluorescence staining results showed that the positive labeling cells of GAP-43 in AG1478 group were more than that of injury control group (P< 0.01). Western blot results showed that the level of GAP-43 protein expression was significant higher in AG1478 group than that in the control group or sham group (P< 0.01). Hindlimb of rats in injury control group and AG1478-treated group were almost totally paralysed at first day post injury, which indicated that the two group rats underwent the same degree injury. Statistical analysis by repeated-measurement ANOVA indicated that the BBB scores of AG1478 group rats were significantly higher than that of control group rats. And statistical analysis by repeated-measurement ANOVA indicated that the body weight of AG1478 group rats were significantly higher than that of control group rats. (P<0.05).Conclusion Acute local administration of epidermal growth factory receptor inhibitors AG1478 in the contusion site could attenuate locomotor function impairment in spinal cord injured rats and significantly upregulated the level of GAP-43 expression. The robust effects of epidermal growth factor receptor inhibitors AG1478 on spinal cord injury indicated that epidermal growth factor receptor inhibitors could be efficient protective agent on central nervous system injury. Part two Effects of epidermal growth factor receptor inhibitor AG1478 on reactive astrogliosis after spinal cord injury in ratsObjective Astrogliosis are thought to play a key role in the pathophysiology of spinal cord injury (SCI), and there are a close relationship between astrogliosis and the activation of epidermal growth factor receptor in the pathophysiology of central nervous system injury. We sought to administer epidermal growth factor recepter AG1478 to treat spinal cord injury in order to investigate the effects of epidermal growth factor receptor inhibitor AG1478 on the expression of pEGFR in astrocytes and reactive astrogliosis and production of axon growth inhibitory factor chondroitin sulphate proteoglycans after rat spinal cord injury.Methods The rats spinal cord injury model were built by using the weight-drop method, and sham-operated animals underwent T10 laminectomy alone. Forty five SD health female adult rats were randomly divided into 3 equal groups:sham group, injury control group and AG1478-treated group. The rats in AG1478-treated group were locally administered 0.2 ml 2mM AG 1478 using a Gelfoam as a carrier, while the sham group and control group were locally administered dimethylsulfoxid also using a Gelfoam as a carrier. All rats were sacrificed at 14d and 28d. The expression of GFAP at day 14 and day 28 after spinal cord injury were detected by immunofluorescence and western blot. The expression of phosphorylated epidermal growth factor receptor inhibitors in astrocytes at day 14 after spinal cord injury was observed by double-immunofluorescence and the expression of chondroitin sulphate proteoglycans in astrocytes at day 28 after spinal cord injury were observed by double-immunofluorescence.Results GFAP-positive cells were sparse and slim in the sham group. In contrast, activated astrocytes characterized by the hypertrophy of astrocytic processes and up-regulation of GFAP were present at 2 weeks after SCI and the typical glial scar became evident at 4 weeks after SCI in the control group. While the up-regulation GFAP was significantly suppressed at 2 weeks after SCI and the dense glial scar was not observed at 4 weeks after SCI in the AG1478 group. Western blot analysis also showed that the GFAP expression of AG1478-treated group at 2 and 4 weeks after SCI was significant lower than that of control group (P< 0.01). By double-immunofluorescence, positive labeling of pEGFR and GFAP was weakly present or absent in sham group and no colozalition of pEGFR and GFAP appeared in astrocytes in sham group, which indicated that the astrocytes in sham group did not expressed pEGFR. While positive labeling of pEGFR and GFAP was strongly present in control group. The cyst and glial scar were formed at the lesion center in control group, and there was significant colozalition of pEGFR and GFAP in the glia scar around the cyst. This indicated that astrocytes after spinal cord injury expressed pEGFR. The immunofluorescence intensity of pEGFR and GFAP in AG1478-treated group was significantly weaker compared with that in control group(P<0.01), which indicated that the expression of pEGFR and GFAP was suppressed by AG1478. No colozalition of pEGFR and GFAP appeared in AG1478-treated group. Positive labeling of CSPGs and GFAP was weakly present in sham group, while positive labeling of CSPGs and GFAP was strongly present in control group. Moreover, there was significant colozalition of CSPGs and GFAP in the glial scar, which indicated that active astrocytes significanlty expressed CSPGs. The immunofluorescence intensity of CSPGs and GFAP in AG1478-treated group was significantly weaker compared with that in control group(P＜0.01), which indicated that the expression of CSPGs and GFAP was suppressed by AG1478.Conclusion Astrocytes were significantly up-regulated the expression of pEGFR after SCI. Epidermal growth factor receptor inhibitor AG1478 can attenuate the reactive astrogliosis and the prodution of inhibitory factor CSPGs after SCI by inhibiting the active pEGFR in astrocytes after spinal cord injury. Part three Effects of epidermal growth factor receptor inhibitor AG1478 on demyelination and the expression of myelin-associated inhibitory molecules after spinal cord injury in ratsObjective Traumatic spinal cord injury (SCI) not only cause damage to neuron and axon but also induces harmful effects on the oligodendrocytes, which contribute to severe demyelination. At the same time, the damage oligodendrocytes release lots of myelin-associated inhibitory molecules, which induce an harmful influence on the axonal degeneration after spinal cord injury. Since epidermal growth factor receptor inhibitor AG1478 has a robust effects on the function recovery of spinal cord injury, we sought to administrate AG1478 to treat spinal cord injury in order to investigate the effects of AG1478 on demyelination and the mRNA expression of myelin-associated inhibitory molecules MAG,Nogo-A and OMgp after rat spinal cord injury.Methods Thirty three health female adult SD rats were randomly divided into 3 equal groups:control group undergoing SCI by weight drop method and acute local administration dimethylsulfoxide using a Gelfoam as a carrier after SCI, AG1478-treated group undergoing SCI by weight drop method and acute local administration 0.2 ml 2mM AG1478 using a Gelfoam as a carrier after SCI, and sham group undergoing T10 laminectomy alone and acute local administration dimethylsulfoxide using a Gelfoam as a carrier postoperation. All rats were sacrificed at 14d and 28d post injury. The demyelination of all group rats at day 28 after spinal cord injury was observed by Luxol Fast Blue staining. The mRNA expression of MAG,Nogo-A and OMgp in all group rats at day 14 and day 28 after spinal cord injury were detected by RT-PCR.Result By Luxol Fast Blue staining, sham-operated spinal cord tissue was characterized by light blue equably across the entire spinal cords, while there was normal coloring deletion area in the lesion center of injury spinal cord tissue, which indicated that demyelination was severe in the lesion center. And the normal coloring deletion area was significantly reduced in the lesion center of AG1478-treated group spinal cord tissues. The demyelination area of AG1478-treated group was significant smaller compared with that of control group(P<0.01). The mRNA expression of myelin-associated inhibitory molecules MAG,Nogo-A and OMgp of sham group was weak post injury, while the mRNA expression of myelin-associated inhibitory molecules MAG,Nogo-A and OMgp of control group significantly increased at day 14 and day 28 after spinal cord injury. The mRNA expression level of myelin-associated inhibitory molecules MAG,Nogo-A and OMgp of AG1478-treated group was significant lower compared to that of control group (P<0.01).Conclusion Local administration of epidermal growth factor receptor inhibitor AG1478 can attenuate the demyelination after spinal cord injury and reduces the mRNA expression level of myelin-associated inhibitory molecules MAG,Nogo-A and OMgp post injury.