Regulatory Effects Of BDNF And It's Pro-protein Following Inflammatory Pain In Rats

Part I Inflammatory pain induced ProBDNF and its receptors expression change in spinal cord and DRG of ratsObjective:To explore expression changes of ProBDNF and its receptors P75NTR and Sortilin in the spinal cord and dorsal root ganglion (DRG) followed by Complete Freund's Adjuvant (CFA) induced inflammatory pain.Methods:Rats were received plantar subcutaneous injection of CFA and saline mixed solvent100ul, establishing inflammatory pain model. The experimental animals were survived1d,7d and14d after injection of CFA respectively. Normal rats served as normal control. The change of50%paw withdrawal threshold (PWT) was observed at1h,6h after CFA injection and the following1d,7d and14d by Von Frey fibers, while the expressions of ProBDNF, P75NTR and Sortilin in spinal cord and DRG were detected by immunohistochemistry and Western blot.Results:1.50%PWT decreased quickly at1h after CFA injection, and reached the lowest value at6h-1d.50%PWT gradually rose at3d and was still lower than baseline at14d.2. Compared with the contralateral side, ProBDNF expression was significantly increased in ispilateral spinal dorsal horn at1d after CFA injection. The increase continued until7d, and gradually restored at14d but still higher than the control group. Western blot showed that ProBDNF expression in ispilateral spinal cord was upregulated at7d after CFA infusion and was significantly more than the contralateral side. The results of DRG immunohistochemistry and Western blot showed that at1d after CFA infusion, expression of ProBDNF in the ispilateral DRG was increased, which last until the7d. ProBDNF was firstly found expressed in the small diameter cells at CFA1d, and then gradually in the medium and large diameter cells at CFA7d. In comparison to CFA1d, the expression of ProBDNF at CFA7d continued to be much higher, and with significantly increased in ipsilateral than the contralateral side. Then the expression of ProBDNF was reduced at14d after CFA infusion and had no difference with normal group.3. The results of spinal cord Immunohistochemistry and Western blot demonstrated that at1d after the CFA infusion, the expression of P75NTR in spinal dosal horn reached summit and maintained to7d, but there was no difference between the ispilateral side and contralateral side. At14d, it displayed a downward expression of P75NTR, but still higher than the control group. The results of DRG Immunohistochemistry and Western blot indicated that at1d after the CFA infusion, the expression of P75NTR was very strong and was higher than the control group, but there was no difference between the left side and right side. The expression of P75NTR was still higher at7d after CFA infusion, which showed no difference to CFA1d. It displayed a downward expression of P75NTR at14d.4. Sortilin positive products were weak in the normal superficial spinal dorsal horn. There was no significant difference of Sortilin expression in the spinal dorsal horn and DRG at different time points after CFA injection. It was worth noticing that in the DRG, Sortilin protein was firstly found expressed in the small diameter cells, and then gradually in the medium and large diameter cells, which was similar to the proBDNF expression.Conclusions:CFA can induce inflammatory pain more than2weeks. ProBDNF and P75NTR upregulation in spinal dorsal horn and DRG may be related to peripheral pain signal transmission and central sensitization in inflammatory pain. Sortilin was involved in the transportation and release of ProBDNF and BDNF probably. Part â…¡ Inflammatory pain induced different BDNF Exons expression change in spinal cord and DRG of ratsObjective:To detect total mRNA of BDNF and different BDNF exons expression change in spinal cord and DRG followed by Complete Freund's Adjuvant (CFA) induced inflammatory pain and reveal differential mechanisms of BDNF gene transcription in inflammatory nociception processes.Methods:Rats were received plantar subcutaneous injection of CFA and saline mixed solvent100ul, establishing inflammatory pain model. The experimental animals were survived1d,7d and14d after injection of CFA respectively. Normal rats served as normal control. The expressions of total BDNF mRNA and different BDNF exons in spinal cord and DRG were detected using RT-PCR and realtime RT-PCR.Results:1. At1d after CFA infusion, there was a significant increase in the expression of BDNF mRNA in ispilateral spinal cord, and also higher than the contralateral side. The upregulation reached the top at1d, and also increased in contralateral side. The expression of BDNF mRNA was reduced at14d, which was still higher than that of the control group. There was a significant increase in the expression of BDNF mRNA in ispilateral DRG at1d after CFA injection and was higher than the contralateral side. The upregulation was last to7d after CFA infusion, and then reduced to normal level at14d.2. Exon â…  was significantly increased in the ipsilateral spinal cord at1d after CFA treatment compared to the contralateral side, and meanwhile exon â…¡A and exon IXA were higher in contralateral side. At CFA7d, exon â…¡A, exon â…¡B and exon â…¨A in ipsilateral spinal cord were up-regulated compared to the contralateral side. Exon â…¡B was still enhanced in bilateral spinal cord at14d after CFA infusion and the increase of exon â…¡A and exon â…¨A were seen in contralateral side.3. The expression of exon â… , exon â…¡A, exon â…¡B and exon â…£ in ipsilateral DRG was significantly increased at1d after CFA infusion, which was significantly higher than the contralateral side. Exon â…  expression was significantly still higher in ipsilateral DRG at CFA7d compared to CFA1d, and also stronger than the contralateral side, other exons expression had no obvious changes. Expressions of all exons were similar to normal rats in DRG at14d after CFA injection.Conclusions:Differential mechanisms of BDNF gene transcription were seen in different parts and different time during inflammatory pain. The change of BDNF exons is coincident with the results of total BDNF mRNA. Part â…¢ Down-regulation of spinal acetylated histone3in the CFA-induced inflammatory pain in rats:Effect of morphineObjective:To explore expression changes of acetylated histone3(ACH3) in spinal dorsal horn followed by Complete Freund's Adjuvant(CFA) induced inflammatory pain and discuss its involvement in nociception processes and effects of morphine.Methods:Rats were received plantar subcutaneous injection of CFA and saline mixed solvent100ul, establishing inflammatory pain model. The experimental animals were survived1d,7d and14d after injection of CFA. Morphine intervention was administrated by intraperitoneal injection (10mg/kg). Normal rats served as normal control. The expressions of ACH3and BDNF and the cell type of ACH3in spinal dorsal horn were detected using immunohistochemistry and double labeling immunofluorescence. The effect of morphine on50%PWT was measured using Von Frey fibers.Results:1. At1day after CFA injection, the expression of ACH3was reduced in the spinal dorsal horn as determined by immunohistochemistry. Until day7post-injection, the expression of ACH3was recoveried. Double labeling immunofluorescence showed that ACH3was expressed mainly in the neurons. A small number of glial cells also expressed ACH3. ACH3expression in neurons was decreased at1day after CFA injection. The decreased ACH3was recovered at day7post-injection. In addition to the recovery expression in neurons, extensive ACH3positive staining was also co-localized in the microglia and astrocytes.2. Morphine administration enhanced50%PWT at1day after CFA injection significantly and attenuated the down-regulated ACH3in the spinal cord. Morphine affected ACH3positive neurons and glial cells, especially astrocytes and microglia cells.3.There was weakly positive expression of BDNF in spinal dorsal horn of normal control group, which increased in ipsilateral side at1d after CFA injection. However, morphine had no effect on BDNF expression in dorsal horn of spinal cord at CFA1d.Conclusions:These results suggest that inflammatory pain induced transient down-regulation of ACH3in the spinal dorsal horn which can be reversed by morphine. There was a phenotype shift of the ACH3expression in the development of inflammatory pain. Morphine has no effect on the expression of BDNF in the spinal dorsal horn in the early phase of inflammatory pain.