Effect of nerve growth factor gene injection on the nerve regeneration in rat lingual nerve crush-injury model

Gao, En Feng; Chung, Hun Jong; Ahn, Kang Min; Kim, Soung Min; Kim, Yun Hee; Jahng, Jeong won; Lee, Jong Ho

Abstract

PURPOSE: Lingual nerve (LN) damage may be caused by either tumor resection or injury such as wisdom tooth extraction, Although autologous nerve graft is sometimes used to repair the damaged nerve, it has the disadvantage of necessity of another operation for nerve harvesting. Moreover, the results of nerve grafting is not satisfactory. The nerve growth factor (NGF) is well-known to play a critical role in peripheral nerve regeneration and its local delivery to the injured nerve has been continuously tried to enhance nerve regeneration. However, its application has limitations like repeated administration due to short half life of 30 minutes and an in vivo delivery model must allow for direct and local delivery. The aim of this study was to construct a well-functioning rhNGF-beta adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with enhanced and extended secretion of hNGF from the injured nerve by injecting rhNGF-beta gene directly into crush-injured LN in rat model.
MATERIALS AND METHODS
hNGF-beta gene was prepared from fetal brain cDNA library and cloned into E1/E3 deleted adenoviral vector which contains green fluorescence protein (GFP) gene as a reporter. After large scale production and purification of rhNGF-beta adenovirus, transfection efficiency and its expression at various cells (primary cultured Schwann cells, HEK293 cells, Schwann cell lines, NIH3T3 and CRH cells) were evaluated by fluorescent microscopy, RT-PCR, ELISA, immunocytochemistry. Furthermore, the function of rhNGF-beta, which was secreted from various cells infected with rhNGF-beta adenovirus, was evaluated using neuritogenesis of PC-12 cells. For in vivo evaluation of efficacy of rhNGF-beta adenovirus, the LNs of 8-week old rats were exposed and crush-injured with a small hemostat for 10 seconds. After the injury, rhNGF-beta adenovirus(2 microliter, 1.5 x 10(11)pfu) or saline was administered into the crushed site in the experimental (n=24) and the control group (n=24), respectively. Sham operation of another group of rats (n=9) was performed without administration of either saline or adenovirus. The taste recovery and the change of fungiform papilla were studied at 1, 2, 3 and 4 weeks. Each of the 6 animals was tested with different solutions (0.1M NaCl, 0.1M sucrose, 0.01M QHCl, or 0.01M HCl) by two-bottle test paradigm and the number of papilla was counted using SEM picture of tongue dorsum. LN was explored at the same interval as taste study and evaluated electrophysiologically (peak voltage and nerve conduction velocity) and histomorphometrically (axon count, myelin thickness).
RESULTS
The recombinant adenovirus vector carrying rhNGF-beta was constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. GFP expression was observed in 90% of rhNGF-beta adenovirus infected cells compared with uninfected cells. Total mRNA isolated from rhNGF-beta adenovirus infected cells showed strong RT-PCR band, however uninfected or LacZ recombinant adenovirus infected cells did not. NGF quantification by ELISA showed a maximal release of 18865.4+/-310.9pg/ml NGF at the 4th day and stably continued till 14 days by rhNGF-beta adenovirus infected Schwann cells. PC-12 cells exposed to media with rhNGF-beta adenovirus infected Schwann cell revealed at the same level of neuriteextension as the commercial NGF did. rhNGF-beta adenovirus injected experimental groups in comparison to the control group exhibited different taste preference ratio. Salty, sweet and sour taste preference ratio were significantly different after 2 weeks from the beginning of the experiment, which were similar to the sham group, but not to the control group. Bitter test showed noticeable difference in the preference ratio at week 1, 2, 3, and then all taste preference ratio were normalized after 4th week. After LN injury, the numbers of fungiform papillae were rapidly reduced with the tendency of slow increase from the 3rd week. The conduction velocity was slowly increased both in rhNGF-beta adenovirus and saline injected group within four weeks with the highest peak voltage, appearing during stimulation after 2 weeks, and then gradually reduced in the rhNGF-beta adenovirus injected group at week 1 and 2. Histomorphometric analysis revealed significant increase in axon numbers and myelin thickness in the experimental group.
CONCLUSION
The construction of rhNGF-beta adenovirus and the induction of over-expression of bioactive NGF were successful in both non-neuronal and neuronal cells. rhNGF-beta adenovirus injected into the crush-injured LN induced significant amelioration of nerve regeneration and taste discrimination ability compared to the control group. rhNGF-beta adenovirus injection may be an efficient way to introduce NGF to the injured peripheral nerve in paradigms of research or therapy.