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Hepatocyte Growth Factor Incorporated Into Herpes Simplex Virus Vector Accelerates Facial Nerve Regeneration After Crush Injury

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Hepatocyte Growth Factor Incorporated Into Herpes Simplex Virus Vector Accelerates Facial Nerve Regeneration After Crush Injury Gene Therapy (2011) 18, 1063–1069 & 2011 Macmillan Publishers Limited All rights reserved 0969-7128/11 www.nature.com/gt ORIGINAL ARTICLE Hepatocyte growth factor incorporated into herpes simplex virus vector accelerates facial nerve regeneration after crush injury S Esaki1,2, J Kitoh3, S Katsumi1, F Goshima2, H Kimura2, M Safwat1, K Yamano1, N Watanabe1, N Nonoguchi4, T Nakamura5, RS Coffin6, S-I Miyatake4, Y Nishiyama2 and S Murakami1 Hepatocyte growth factor (HGF) promotes regeneration of the central nervous system, but its effects on the peripheral nervous system remain unclear. This study was conducted to elucidate the effect of HGF on regeneration of the murine facial nerve after crush injury. To do so, a replication-defective herpes simplex virus vector that incorporated HGF was prepared (HSV-HGF). The main trunk of the facial nerve was compressed by mosquito hemostats, and HSV-HGF, control vector or medium was then applied to the compressed nerve. We found that mice in the HGF group required significantly fewer days for complete recovery from nerve compression. Furthermore, the amplitude of the evoked buccinator muscle compound action potential increased following HSV-HGF application. HGF expression in and around the compressed nerve was demonstrated by enzyme-linked immunoassay and immunohistochemistry. In addition, HSV-HGF introduction around the damaged nerve significantly accelerated recovery of function of the facial nerve. These data suggest a possible role of HGF in promoting facial nerve regeneration after nerve damage. Furthermore, this viral delivery method may be applied clinically for many types of severe facial palsy during facial nerve decompression surgery. Gene Therapy (2011) 18, 1063–1069; doi:10.1038/gt.2011.57; published online 12 May 2011 Keywords: facial nerve injury; hepatocyte growth factor; HSV INTRODUCTION promote peripheral nerve regeneration; however, little is known about Numerous studies have used the facial nerve axotomy model to its effects on the peripheral nerve systems. investigate the survival-promoting effects of neurotrophic and growth Herpes simplex virus (HSV) type 1 vectors have been widely used factors on axotomized motor neurons. Ciliary neurotrophic factor was for gene transfer of neurotrophic factors24–28 because they are capable the first factor to demonstrate motor neuronal survival following local of infecting the cells of the nervous system. However, an inherent application to lesioned axons in the newborn rat.1 Subsequently, other difficulty with HSV as a vector system lies in disabling the virus from neurotrophic and growth factors were found to prevent axotomy- being neurotoxic while maintaining its infection efficiency. To address induced motor neuron cell death, including neurotrophin-4/5,2 this problem, we made a replication-defective HSV type 1 vector in insulin-like growth factor-1,3 leukemia inhibitor factor,3 brain-derived which replication was disabled by deleting three critical genes for viral neurotrophic factor,4 nerve growth factor,5 neurotrophin-3 (ref. 6) replication.29–31 The availability of this vector has already been and glial cell line-derived growth factor.7 Although these neurotrophic examined in the nervous system.30–32 factors have been shown to promote regeneration of the injured nerve, In the present study, we incorporated HGF into the replication- the functional outcomes have often been limited.8,9 defective HSV vector and inoculated murine facial nerves with the Hepatocyte growth factor (HGF) is a disulfide-linked heterodimeric vector after compression. Here, for the first time, we demonstrate that protein that was initially purified and cloned as a potent mitogen for HGF prevents peripheral nerve degeneration and promotes regenera- hepatocytes10,11 and a natural ligand for the c-Met protooncogene tion of facial nerve function in a murine model of crush injury. product.12 Subsequently, several functions have been ascribed to HGF, including anti-apoptosis, angiogenesis, motogenesis, morphogenesis, RESULTS hematopoiesis, tissue regeneration in a variety of organs and enhance- Time course of facial nerve paralysis after compression ment of neurite outgrowth.13–20 Previous studies have reported that Facial nerve paralysis developed immediately after compression in all HGF protected neurons from damage and enhanced regeneration in mice and then gradually improved and recovered completely by day the central nervous system in vivo.13,19–23 Given the known effects in 14. The time courses of facial scores for mice in each group are shown the literature, HGF is thought to protect neurons from damage and in Figure 1a. There was no significant difference in final facial scores 1Department of Otolaryngology, Head and Neck Surgery, Nagoya City University Graduate School of Medical Sciences and Medical School, Nagoya, Japan; 2Department of Virology, Graduate School of Medicine, Nagoya University, Nagoya, Japan; 3Professor Emeritus of Nagoya University School of Medicine, Nagoya, Japan; 4Department of Neurosurgery, Osaka Medical College, Osaka, Japan; 5Division of Molecular Regenerative Medicine, Osaka University Graduate School of Medicine, Osaka, Japan and 6Department of Molecular Pathology in Windeyer Institute of Medical Sciences of University College, London, UK Correspondence: Dr S Esaki, Department of Otolaryngology, Head and Neck Surgery, Nagoya City University Graduate School of Medical Sciences and Medical School, Kawasumi 1, Mizuho-ku, Nagoya 467-8602, Japan. E-mail: [email protected] Received 13 August 2010; revised 19 January 2011; accepted 24 February 2011; published online 12 May 2011 HGF-HSV accelerates regeneration of facial nerve SEsakiet al 1064 6 HGF 15 ## $$ 5 $$ ##** 12 4 ##** ##** 9 3 PBS 2 6 Facial Nerve Score ##** LacZ 1 Days for complete recovery 3 *,** LacZ vs. HGF #* #* #,## PBS vs. HGF 0 0 0 1 2 34567891011121314 HGF LacZ PBS days after operation Figure 1 HSV-HGF enhances recovery after crush injury. (a) Time course of facial nerve scores in the HGF (n¼5), LacZ (n¼5) and PBS (n¼15) groups. Error bars indicate standard deviation. Mice in the LacZ and PBS groups showed almost the same degree of recovery, whereas mice in the HGF group showed significantly improved recovery (*,#Po0.05; **,##Po0.01). (b) Days required for complete recovery. Mice in the HGF group required significantly fewer days to recover ($$Po0.01). Error bars indicate standard deviation. 3 14 2.5 12 HGF 2 10 HGF ** 1.5 LacZ 8 ** ** 6 LacZ ** 1 4 ** 0.5 CMAP Amplitude (mV) 2 HGF concentration (ng/nerve) 0 0 Pre- Day7 Day14 Pre- Day3Day7 Day14 operation operation Figure 2 (a) Evoked buccinator muscle CMAP amplitude also showed accelerated recovery. Mean amplitude for the HGF group was significantly greater than that for the LacZ groups on days 7 and 14 (**Po0.01). Error bars indicate standard deviation. (b) HGF was detected from the extracts of murine facial nerve. The amount of HGF in the HGF group was greater than that in the LacZ group on days 3, 7 and 14 (**Po0.01). Error bars indicate standard deviation. on day 14 between mice in the LacZ and PBS groups. However, the amplitude was significantly larger (Po0.01, unpaired t-test) for the facial scores for mice in the HGF group on days 7–12 were signifi- LacZ group than the HGF group. However, CMAP amplitudes cantly higher than the scores for mice in the PBS and LacZ groups. We increased between days 7 and 14 in both the LacZ and HGF groups. compared the number of days required to reach a score of 6, which On day 14, the CMAP amplitude was 6.3±0.3 mV (53%) in the LacZ indicates complete recovery from facial paralysis. The PBS group group and 8.2±0.6 mV (70%) in the HGF group. Furthermore, the required 11.5±0.9 days, the LacZ group required 11.6±1.0 days and HGF group showed significantly greater recovery of CMAP amplitude the HGF group required 10.3±0.5 days (Figure 1b). Recovery from compared with the LacZ group (Po0.01, unpaired t-test). Finally, the facial nerve paralysis in the HGF group was significantly faster than in difference in CMAP amplitudes between the HGF group and the LacZ either the LacZ or the PBS groups (Po0.01, unpaired t-test). No mice group was larger on day 14 than on day 7. died or showed any significant weight loss or other complications during this experiment. HGF secretion in nerve extracts The amount of HGF in the facial nerve before surgery was Electrophysiological examination 1.62±0.11 ng (Figure 2b). HGF in the LacZ group increased to The mean evoked compound muscle action potentials (CMAPs) 2.03±0.95 ng on day 3 following surgery, then decreased to amplitude of the buccinator muscle was 11.8±1.1 mV in both groups 1.56±0.09 ng on day 7. HGF levels showed no significant differences combined before nerve compression. On day 7, CMAP amplitude on days 7–14 (1.59±0.11 ng measured on day 14). On the other hand, decreased to 5.2±0.3 mV (44%) in the LacZ group and 6.2±0.6 mV the HGF level in the HGF group increased to 2.31±0.16 ng on day 3 (53%) in the HGF group (Figure 2a). The decrease in CMAP and then decreased slightly to 2.08±0.21 ng on day 7. This level was Gene Therapy HGF-HSV accelerates regeneration of facial nerve SEsakiet al 1065 maintained until day 14 (2.11±0.14 ng). Furthermore, HGF levels in regenerating nerve fibers were seen on days 7 and 14. Furthermore, on the HGF group were significantly higher than levels in the LacZ group days 7 (Figures 5b and e) and 14 (Figures 5c and f), the number of between days 3 and 14 (Po0.01, unpaired t-test). myelinated nerve fibers was greater in the HGF group (276±58.6 fibers per mm2 on day 7 and 298±53.6 fibers per mm2 on day 14) Expression of reporter protein (KT3) in the facial nerve’s than in the LacZ group (164±24.9 fibers per mm2 on day 7 and compressed region and nucleus 218±47.3 fibers per mm2 on day 14).
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