The Role And Mechanism Of ProBDNF In Incisional Pain After Hind Paw Incision In Rats

Chapter I:Expression of proBDNF in the dorsal root ganglia and its role in incisional pain after hind paw incision in rats.ObjectiveTo investigate the changes of proBDNF expression in the dorsal root ganglia after incision and the effect of anti-proBDNF in the surgical incision-evoked pain hypersensitivity.MethodsIncisional pain rat model was established by a longitudinal incision in right plantar hind paw.under isoflurane anesthesia. Dorsal root ganglias were removed at various post-operative times (1-71h). Expression pattern of proBDNF was determined by immunofluorescence. Anti-proBDNF was administered intraperitoneal before incision and paw withdrawal threshold (PWT) was measured at the indicated time after incision.ResultsThe expression of proBDNF in the ipsilateral lumbar DRG was increased after hind-paw incision within1-24h. Hind paw incision dramatically decreased PWT after surgery. However, the decreased PWT induced by hind paw incision was obviously attenuated by anti-proBDNF pretreatment through i.p injection.ConclusionThe present study showed that incision induced the upregulation of proBDNF in the dorsal root ganglia and the upregulated proBDNF contributed to the pain hypersentivity induced by surgical incision. Chapter II Construction and identification of adenovirus mediated proBDNF geneObjectiveTo construct adenovirus mediated proBDNF gene for the delivery in tissue in vivo.MethodsproBDNF gene was amplified from the pcDNA3.1plasmid encoding proBDNF gene using the PCR method. The pDC315-GFP vector was digested by restriction enzyme, and PCR products of proBDNF were recovered by using electrophoresis, then liagated into the pDC315-GRP. Thereafter, the vector encoding proBDNF gene were transformed into bacterial competent cells. The colonies were picked up and identified by PCR, then sequenced and analyzed by comparative analysis. After confirming the right colonies by sequencing, the colonies were transfected into293T cells for24hours using Lipofectamine2000. The expression of GFP were observed by fluorescence microscopy. Well-grown purpose cells were collected and protein was extracted. The expression of target genes were detected by Western blot. The recombinant pDC315-proBDNF adenovirus was prepared by using AdMax adenovirus packaging system, including the adenovirus shuttle plasmid and secondary packaging plasmid (pBHG lox ΔE1,3Cre). The taget virus was transfected into the HEK293cells. The virus were then amplified, purified and identified by Western Blot.ResultsThe recombinant pDC315-proBDNF adenovirus was generated by homologous and identified by PCR and Western Blot. The proBDNF gene fragment size was741bp. The primary virus titer was8.0×10100PFU/ml, proBDNF was expressed in HEK293cells effectively.ConclusionThe proBDNF adenovirus vector was constructed successfully. Chapter III:The effect of overexpression of proBDNF in the hind paw on paw withdrawal threshlodObjectiveTo investigate the pain threshold and morphological changes after overexpression of proBDNF through plantar injection of proBDNF adenovirus vector.MethodsSixteen rats were randomly divided into two groups:blank adenovirus vector group (n=8) and proBDNF adenovirus vector group (n=8), plantar injection of blank adenovirus vectors and proBDNF adeno-viral vectors (109pfu) respectively. Before injection and3days after injection, PWT was detected after foot mechanical stimulation. The tissue around the injecting site was dissected and harvested at day3after injection. Expression of proBDNF was detected by Western blot,fluorescence microscope,immunofluorescence staining and immunohistochemistry. Hematoxylin and Eosin staining was performed to examine the morphological changes in response to proBDNF adenovirus injection.Results1) ProBDNF adenovirus vector was highly expressed in the rat foot, the overexpressed proBDNF was mainly localized in the nerve fibers.2) A large number of inflammatory cells were infiltrated in the subcutaneous tissue at day3after proBDNF adenovirus vector injection and obvious swelling at the injection site.3) Three days after injection, compared with the control adenovirus vector, PWT was significantly reduced in the adenovirus mediated proBDNF gene group.ConclusionProBDNF adenovirus vector was highly expressed in nerve fibers after hind paw injection; Overexpression of proBDNF in the hind paw resulted in mechanical hypersensitivity as indicated by the decreased PWT and tissue inflammation. Thus, proBDNF contributed to pain hypersensitivity along with tissue inflammation suggesting that proBDNF may be a novel target for surgical pain management.