The Effect Of Anti-ProBDNF Combined With BDNF Over-expression On Spinal Cord Injury In Mice

Objective:To investigate the role of proBDNF blockage combined with BDNF over-expression on the recovery of SCI in BDNF transgenic mice and explore the underlying mechanism so as to find the useful experimental evidence with regard to treatment of spinal cord injury.Method1. To construct BDNF overexpression transgenic mouse model:BDNF cDNA was inserted to the downstream of promoter systematically expressing CMV in order to construct the vector that expressed target gene (BDNF). Microinjection was used to inject the transgenic vector into the fertilized ovum of C57BL/6J mice. So, the CMV-BDNF transgenic mouse model was constructed successfully. The genotypes of transgenic mice were identified by using RT-PCR. BDNF expression level was ascertained by RT-PCR and ELISA. Immunohistochemitry was employed to detect the subcellular localization of BDNF gene encoding protein in the spinal cord tissue.2. Preparation of spinal cord transected (SCT) mouse models and blockage of ProBDNF antibody:A total of32adult healthy female BDNF over-expression transgenic mice and wild type mice, weighing18to25g, were randomized into four group:sham operated group, control group, ProBDNF antibody blockage group, BDNF overexpression group and BDNF overexpression+ProBDNF antibody blockage group, with12mice in each group. Spinal cord of mice was transected at T10level. The ProBDNF antibody diluted by NS was injected intraperitoneally (10ug/ul) at1,4,8and12d respectively. The injected dose was10ul/g on1the1st day, and a half dose was used at4,8and12d.NS substituted for the ProBDNF antibody in control and BDNF over-expression group.3. Neurobehavioral test:(1)BBB Score Rating Scale was used to evaluate the motor function in hind limbs of mice in each group, once for every two days.(2)Sensory function test:tail-flick test was carried out in mice of each group one month post of operation.4. Measurement of nerve regeneration:One month post of operation, sample harvesting was conducted in3mice in each group. IHC of NeuN, GAP-43,5-HT and GFAP was performed to detect the circumstance of the nerve regeneration.5. Western blot was employed to detect the changes of AKT and ERK protein content and corresponding phosphorylated protein-PAKT and PERK in spinal tissues1-2segment above and below the injury site.6. Statistical analysisResults:1. BDNF over-expression transgenic mice model was successfully constructed. Sample No26held the highest BDNF expression rate in that BDNF exhibited a markedly higher BDNF expression in spinal cord, muscles and cerebral cortex relative to that of wild type one.2. BBB Scores in each group exhibited gradually elevated over time until on the14d post of operation it attained a high peak (P<0.05). During21to28d post of operation, there was significant difference observed among all the four groups one another. Moreover, the BBB Scores in BDNF over-expression group marked higher compared to ProBDNF antibody blockage group (P<0.05), and that of BDNF overexpression+ProBDNF antibody blockage group was prominently higher(p<0.05) relative to BDNF over-expression alone one.3. The time of tai-flick in each group1m after injury showed significantly shortened relative to sham operation group. This time course in control group was prolonged relative to BDNF over-expression group (P<0.05), and had no significant difference with the other groups (P>0.05). There was no marked difference for this time course in ProBDNF antibody blockage group with the other groups. The time course of tail-flick in BDNF over-expression group was marked shortened relative to that of BDNF over-expression+ProBDNF antibody blockage one (P<0.05)4.Immunostaining revealed that the morphological structure of spinal cord in sham operated group was normal in that the cell bodies of neurons were plump with clear processes. The number of NeuN neurons and GAP-43,5-HT positive fibers in the anterior horn of rostral side of injured spinal cord were significantly attenuated relative to sham operated group. While in the ProBDNF antibody blockage group, the number of NeuN neurons and GAP-43,5-HT positive fibers in the anterior horn of rostral side of injured spinal cord was increased relative to the SCI control (P<0.05), especially in the BDNF over-expression+ProBDNF antibody blockage, with BDNF over-expression group the second, ProBDNF antibody blockage group the third. There was significant different for this number in BDNF over-expression+ProBDNF antibody blockage relative to every other group. The number of GFAP positive cells in the anterior horn of spinal cord in SCI control group following SCT was markedly increased relative to the normal group (P<0.01), but had no significant difference compared with BDNF over-expression and BDNF over-expression+ProBDNF antibody blockage group. The number of GFAP positive cells in the anterior horn of spinal cord in BDNF over-expression+ProBDNF antibody blockage group was obviously increased relative to BDNF over-expression group (P<0.01)5. Revealed by Western blot, ERK expression significantly up regulated in the spinal cord1-2segment above and below the injury site in BDNF over-expression+ProBDNF antibody blockage group relative to sham operated group (P<0.01). The level of P-ERK in BDNF over-expression+ProBDNF antibody blockage group showed significantly elevated relative to sham operated group (P<0.05). AKT level in BDNF over-expression+ProBDNF antibody blockage group was much higher than that of sham operated, control and BDNF over-expression group (P<0.05). P-AKT level in BDNF over-expression+ProBDNF antibody blockage group was much higher than that of sham operated group(P<0.05)Conclusions:1. BDNF over-expression transgenic mouse model was constructed successfully, as well as the SCT mice model.2. ProBDNF antibody blockage, combining with BDNF over-expression promoted the amelioration of motor and sensory function, as well as the neuronal survival and nerve fiber regeneration in SCT mouse models.3. ProBDNF antibody blockage, combining with BDNF over-expression did improve the neurobehaviour and neuroplasticity in SCT mice, maybe depend on the possible mechanism involved in ERK (known as an important cellular apoptotic signaling pathway) blockage by means of BDNF deletion, thereby promote the AKT signaling pathway responsible for cellular proliferation and regeneration.