| Traumatic brain injury (TBI) is one of the commonly and frequently encountered diseases,whose morality and mutilation rate are high and lack of ideal therapeutic way still ,which are threatening the life and physical fitness of our human beings. Recent studies demonstrate that rhEPO can significantly reduce cerebral edema and enhance functional recovery from stroke in several animal models.Erythropoietin(EPO) had always been regarded as an acid glycoprotein,which is secreted by kidneys and used to regulate the red blood cell .Recent research showed astrocyte secreted EPO by paracrine in the central nervous system (CNS),which can bind the receptor on the adjacent neuron membrance and produce protective effect.In this study,we focus on some of the cellular mechanisms mediating the neuroprotective effects of rhEPO, aiming to learn more about how functional recovery occurs after TBI. Firstly,we established a modified Feeney's TBI rat model and observed the regulation of EPO and EPOR expression after TBI. Secondly, we observed the effects of rhEPO on the morphological and functional recovery. Some indicators related to the pathophysiological process of TBI, including posttraumatic brain edema, improvements neurological recovery were also examined.Thirdly, in order to clarify whether rhEPO exert its neuroprotective effects through the antiapoptosis, we studied the apoptosis of neurons with TUNEL and the expressions of antiapoptotic Protein Bcl-2 and apoptotic protein bax with RT-PCR ,western blot and immunofluorescence after TBI.Part 1 The expression of erythropoietin and its receptor in adult rats brain after acute traumatic brain injuryObjective To investigate the regulation of erythropoietin (EPO) and erythropoietin receptor (EPOR) expression in adult rats brain after acute traumatic brain injury(TBI).Methods TBI models were established by feeney s method..The expressions of EPO and EPOR at respectively time points were detected by reverse transcription polymerase chain reaction (RT-PCR),western blot and immunofluorescence staining.Results EPOmRNA expression sharply increased at 12 h (0.327±0.022) , peaked at 24 h (0.374±0.016) to 3 d (0.491±0.035),returned to normal level at 5 d afterTBI.The EPOR expression as early as 12 h after injury. significantly increased at 24 h (0.736±0.017), sustained to peak from 24 h to 7 d (0.771±0.017). EPO protein expression sharply increased at 24 h(0.46±0.072) , peaked at 3 d (0.58±0.063),returned to normal level at 7 d after TBI.The EPOR protein significantly increased at 24 h (0.83±0.073), sustained to peak from 24 h to 7 d (0.71±0.075). Conclusion The EPOR expression can have being up-regulated significantly during about 7 days after TBI while the EPO is only shortly elevanted.The protective mechanism of EPO on central nerve is possibly related to the marked increasing of erythropoietin receptor expression after TBI.Part 2 Effects of rhEPO improve neurological function after experimental traumatic brain injuryobjective Effects of rhEPO improve neurological function after experimental traumatic brain injury.Methods 130 male Wistar rats , weighing280-320g, were randomly divided into 3 groups:rhEPO treatment group(treated with EPO 5000 IU/kg ip once a day following TBI), TBI group (treated with normal saline 30 mg/kg ip once a day following TBI ) ,sham operation group. A battery of behavioral tests was performed at 24 h, 3,5 and 7d after TBI based on the modified Neurological Severity Score (mNSS). Brain water content was determined by the dry-wet weight method. Sections were used for HE staining and NISS staining.Results the mean brain water content was lower (78.15±0.56)in the EPO group than the control group(84.25±0.37) at 24 hours after TBI. After 72 hours of TBI,the brain water content increased,the mean brain water content was 85.58±0.36 in the TBI group and 79.57±0.67 in the rhEPO group. rhEPO group decreased the brain water content distinctive1y compared with the TBI group (P<0.05). H E and NISS staining: 5 hours after TBI,There were lots of neurons disappeared and some red cells appeared in the core of the injury. Most of the neurons were necrosis and edema;some leucocytes infiltrated gently at the edge of injury at 24 hours after TBI. Necrosis appeared distinctly in the TBI group than the rhEPO group at 72 hours,some glia cell surround injured section distinctly increased in the TBI group than the rhEPO group 120 h after TBI. Conclusion The rhEPO distinctly decreased the brain water content after TBI . In a ddition, rhEPO significantly decreased the mNSS scores, improvement neurological function after TBI.It proved that EPO has neuroprotective effects against TBI.Parrt 3 Effect of erythropoietin on the neurocyte apoptosis following traumatic brain injury in ratsObjective To explore the inhibiting effect of recombinant human erythropoietin(rhEPO) on neurocyte apoptosis following traumatic brain injury (TBI)in rats and the related mechanisms.Methods The rats of TBI were induced by Feeney free falling model.130 male Wistar rats were randomly divided into 3 groups: rhEPO treatment group(treated with EPO 5000 IU/kg ip once a day following TBI), TBI group(treated with normal saline 30 mg/kg ip once a day following TBI),sham operation group.Rats were sacrificed at 5 h,12 h,24 h,3 d,5 d,7 d after TBI . The expression of Bcl-2 and Bax gene and protein were detected by reverse transcription polymerase chain reaction (RT-PCR),western blot and immunofluorescence staining after TBI. The apoptosis characteristics of the injured celebral cortex was detected by TUNEL staining.Results①Bcl-2mRNA and protein, mainly concentrated in cell membrane, increased after 24 hours after TBI and bottomed out at 3 d, while the bcl-2 in rhEPO treatment group peaked after 24 h and kept its momentum from 3 d to 7 d.②The bax mRNA level in TBI group reached the peak after 24 h( 0.37±0.052 ), increased gradually from 72 h (0.52±0.051) to 5 d (0.49±0.042) than that in the rhEPO group, which peaked after 24 h(0.22±0.043) and bottomed out at 3 d.( 0.29±0.053)③Tunel staining positive cells appeared at 5 h (25±5.32)after TBI, reaching the peak at 3 d(68±7.51) in the TBI group.The apoptosis cells numbers in the rhEPO treatment group surrounding impact site obviously decreased, compared with the TBI group at respective time points.Conclusions These results indicated that rhEPO treatment Decreases the quantity of the apoptosis neurons,Increases the Expression of antiapoptotic Protein Bcl-2 and decreases the Expression of apoptotic Protein Bax.It may be one of the neuroprotective mechanisms against TBI . |
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