Primary Study Of The Biological Function Of Brain-derived Neurotrophic Factor Precursor

Objective To investigate the biological function of brain-derivedneurotrophic factor precursor(proBDNF) by observing its transportationin peripheral nerves and the possible role on rat retinal ganglion cells(RGCs) after acute high intraocular pressure (HIOP).Methods Sciatic nerves, dorsal roots or dorsal column in adultSprague-Dawley rats were ligated or crushed. After survival for differentperiods of time, injured sciatic nerves, dorsal roots and spinal cord wereexamined for accumulation of endogenous proBDNF around injury sitesby immunohistochemistry or Western blot with specific polyclonalproBDNF antibodies. The proBDNF was detected by immunoelectro-microscopy in the spinal cord and ligated sciatic nerves. PC12 cells werecultured and transformed with the proBDNF plasmid and performed withproBDNF immuno- cytochemistry. Biotinylated or Alexa-488 conjugatedproBDNF or BSA was injected into the sciatic nerve in adult rats. Six orsixteen hours after injection, the rats were perfused and the sciatic nerves,ipsilateral and contralateral L4 and L5 DRG were sectioned, developedand observed under microscope.Adult SD rats were divided into high intraocular pressure withouttreatment(HIOP), proBDNF antiserum pre-treated, normal goat serumpre-treated/HIOP(NGS/HIOP) and proBDNF antiserum pre-treated /HIOP groups. The left eyes of last three groups received intravitrealinjection of NGS or proBDNF antiserum. Two days later, the intraocularpressure of all left eyes of HIOP, NGS/HIOP and proBDNFantiserum/HIOP groups was increased to 102.4mmHg±9.2mmHg, whichmakes b wave of flash electroretinogram disappear, and then maintainedfor 60 minutes. The right eyes served as normal control groups. 1, 3, 7days later, rat retina was stained by Nissl, Hoechst and TUNEL.The hippocampal neurons were dissociated from E17 rats andcultured. Then the cells were treated with proBDNF, preBDNF (pro-peptide of proBDNF) and proBDNF antiserum, respectively. Thirtyminutes, one hour or 48 hours later, the cells were stained with Nisslsolution and ELK-p, ERK2, c-fos immunocytochemistry were performed.Results The immunoreactivity for proBDNF was accumulated atboth proximal and distal segments around the ligature of sciatic nerve,dorsal roots and stumps of cut dorsal column of the spinal cord. Theaccumulation reached maximum 24 hours after injury and lasted at leastfor 7 days at proximal segment and for 3 days at the distal segment.Western blot analysis showed that proBDNF was found in the sciaticnerve segments proximal and distal to the ligature and in the spinal cord.Electromicroscopy showed proBDNF-like immunoreactivity was presentin synaptic vesicles, mitochondria and synapse of primary sensoryneurons in the dorsal horn and large dense-cored vesicles and microtubules in the proximal segment of the ligated sciatic nerves.Pro-BDNF was also detected in the cytoplasm and neurites of PC12 cellstransformed with the pro-BDNF plasmid. After injected into sciatic nerve,biotinylated or Alexa-488 labelled proBDNF was detected along entirelength of sciatic nerve and in dorsal root ganglion neurons.The arrangement of neurons was clear and regular in normal controlgroup by Nissl staining. In NGS/HIOP group, the morphological changeswere similar to that of HIOP group. After HIOP, the cell number and thethickness of inner retina were significant decreased. Compared withNGS/HIOP group, there are some TUNEL positive cells located at theganglion cell layer and the Nissl staining cell number was significantlydecreased, the structure was also disturbed and many Hoechst positivecells were distributed diffusely in the inner retina of proBDNFantiserum/HIOP group. The retinal morphology in proBDNF antiserumtreated group was similar to that in normal control.The expression of ELK-p, ERK2 and c-fos was significantlyupregulated in cultured hippocampal neuron after treated with proBDNFprotein, and sometimes the immunoreactivity was seen in some nucleus.Whereas the expression was downregulated with proBDNF antiserumtreatment, and many cells exhibited swelling and vasoculation. The c-fosimmunoreactivity was upregulated in preBDNF treated cells, but theERK2 and ELK-p immunoreactivity was similar to that in normal cultured cells.Conclusion These results demonstrated that proBDNF wasanterogradely and retrogradely transported in sensory neurons and maybe sorted into vesicles of regulated secretory pathway. It also suggestedthat proBDNF, like mature BDNF, promoted retinal ganglion cellssurvival. Activation of the ERK and ELK pathway may involve in theprotectional mechanism of proBDNF on neuronal survival.