ABSTRACT
BACKGROUND: Several molecular changes occur following axotomy, such as gene up-regulation and down-regulation. In our previous study using Affymetrix arrays, it was found that after the axotomy of sciatic nerve, there were many novel genes with significant expression changes. Among them, neuronatin (Nnat) was the one which expression was significantly up-regulated. Nnat was identified as a gene selectively expressed in neonatal brains and markedly reduced in adult brains. The present study investigated whether the expression of Nnat correlates with symptoms of neuropathic pain in adult rats with transected sciatic nerve. METHODS: Western blotting, immunohistochemistry, and the Randall and Selitto test were used to study the protein content, and subcellular localization of Nnat in correlation with pain-related animal behavior. RESULTS: It was found that after nerve injury, the expression of Nnat was increased in total protein extracts. Unmyelinated C-fiber and thinly myelinated A-delta fiber in adult dorsal root ganglions (DRGs) were the principal sub-population of primary afferent neurons with distributed Nnat. The increased expression of Nnat and its subcellular localization were related to mechanical hyperalgesia. CONCLUSIONS: The results indicated that there was significant correlation between mechanical hyperalgesia in axotomy of sciatic nerve and the increased expression of Nnat in C-fiber and A-delta fiber of adult DRG neurons.
Subject(s)
Ganglia, Spinal/metabolism , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Sciatic Nerve/injuries , Animals , Axotomy , Ganglia, Spinal/physiopathology , Gene Expression , Hyperalgesia/genetics , Hyperalgesia/physiopathology , Immunohistochemistry , Male , Membrane Proteins/genetics , Nerve Fibers, Myelinated/physiology , Nerve Fibers, Unmyelinated/physiology , Nerve Tissue Proteins/genetics , Neuralgia/genetics , Neuralgia/physiopathology , Pain Threshold/physiology , Rats , Rats, Sprague-Dawley , Sciatic Nerve/physiopathologyABSTRACT
BACKGROUND: Gene transfer into many cell types has been successfully used to develop alternative and adjunct approaches to conventional medical treatment. However, effective transfection of postmitotic neurons remains a challenge. The aim of this study was to develop a method for gene transfer into rat primary dorsal root ganglion neurons using sonoporation. METHODS: Dissociated cells from adult rat dorsal root ganglion (DRG) cells were sonicated for 1-8 s at 2.5-10 W to determine the optimal ultrasound duration and power for gene transfection and cell survival. Transfection efficiency was compared between sonoporation, liposome and lentiviral vector gene transfer techniques. RESULTS: The optimum ultrasound intensity was 5 W for 2 s and yielded an efficiency of gene transfection of 31% and a survival rate of 35%. CONCLUSIONS: Sonoporation can be optimized to minimize cell death and yield a high percentage of transfected neurons and that this technique can be easily applied to primary cultures of rat dorsal root ganglion neurons.
