Kazuhisa Moro

Adenoviral GDNF gene transfer prevents motoneuron loss in the nucleus ambiguus.

We examined neuroprotective effects of an adenoviral vector encoding glial cell line-derived neurotrophic factor (AxCAhGDNF) on the lesioned adult rat motoneurons in the nucleus ambiguus. After vagal nerve avulsion, AxCAhGDNF, AxCALacZ (adenovirus encoding beta-galactosidase gene) or PBS was inoculated into the jugular foramen. Four days after the avulsion and treatment with AxCALacZ, the animals expressed beta-galactosidase activity in the lesioned motoneurons in the nucleus ambiguus. The animals avulsed and inoculated with AxCAhGDNF showed immunolabeling for GDNF in the nucleus ambiguus on the treated side and expression of virus-induced human GDNF mRNA transcripts in the brainstem tissue that contained the nucleus ambiguus of the treated side. The treatment with AxCAhGDNF after avulsion prevented the loss of lesioned motoneurons in the nucleus ambiguus, ameliorated the choline acetyltransferase immunoreactivity, and also suppressed the activity of nitric oxide synthase in these neurons. These results indicate that adenovirus-mediated GDNF gene transfer may prevent the degeneration of motoneurons in humans after either vagal nerve injury or recurrent laryngeal nerve injury.

Adenoviral gene transfer of BDNF and GDNF synergistically prevent motoneuron loss in the nucleus ambiguus

We have previously shown that neuroprotective effects of an adenoviral glial cell line-derived neurotrophic factor (GDNF) gene transfer on the lesioned adult rat motoneurons in the nucleus ambiguus. In the present study, we examined neuroprotective effects of adenoviral gene transfer of brain-derived neurotrophic factor (BDNF) or/and GDNF to motoneurons in nucleus ambiguus using an adult rat vagal nerve avulsion model. The animals avulsed and inoculated with adenoviral vectors encoding BDNF (AxCAmBDNFME) or/and GDNF (AxCAhGDNF) showed immunolabeling for BDNF or/and GDNF in the nucleus ambiguus on the treated side, respectively, and expression of virus-induced BDNF or/and GDNF mRNA transcripts in the brainstem tissue that contained the nucleus ambiguus of the treated side. The treatment with AxCAhGDNF or AxCAmBDNFME significantly prevented the loss of vagal motoneurons in comparison to the control; the protective effect of AxCAmBDNFME was greater than that of AxCAhGDNF. The combined treatment with AxCAmBDNFME and AxCAhGDNF acted synergistically and significantly larger number of vagal motoneurons was preserved as compared to either AxCAmBDNFME treatment or AxCAhGDNF treatment. The treatment with AxCAmBDNFME or/and AxCAhGDNF after avulsion also suppressed the activity of nitric oxide synthase in lesioned motoneurons in the nucleus ambiguus. These results indicate that adenovirus-mediated BDNF and GDNF gene transfer may prevent the degeneration of motoneurons in humans after either vagal nerve injury or recurrent laryngeal nerve injury.

Gene therapy for laryngeal paralysis.

Objectives: The surgical options for laryngeal paralysis only achieve static changes of vocal fold position. Laryngeal reinnervation procedures have had little impact on the return of dynamic laryngeal function. The development of a new treatment for laryngeal paralysis, aimed at the return of dynamic function and neurologic restoration and regeneration, is necessary. Methods: To assess the possibility of gene therapy for laryngeal paralysis aiming for the return of dynamic laryngeal function, we investigated the therapeutic effects of gene therapy using rat laryngeal paralysis models. Results: In a rat vagal nerve avulsion model, we transferred glial cell line-derived neurotrophic factor (GDNF) gene into the nucleus ambiguus using an adenovirus vector. Two and 4 weeks after the GDNF gene transfer, a significantly larger number of surviving motoneurons was observed. These neuroprotective effects of GDNF gene transfer were enhanced by simultaneous brain-derived neurotrophic factor gene transfer. In a rat recurrent laryngeal nerve crush model, we transferred GDNF gene into recurrent laryngeal nerve fibers after crush injury. Two and 4 weeks after GDNF gene transfer, we observed significantly faster nerve conduction velocity and better vocal fold motion recovery. Conclusions: These results indicate that gene therapy could be a future treatment strategy for laryngeal paralysis. Further studies will be necessary to demonstrate the safety of the vector before clinical application.

Sentinel node detection in n0 laryngeal cancer

Abstract Problem: Sentinel lymph node (SLN) is the lymph node that directly receives lymphatic flow from a primary cancer lesion. Attention has been focused on the SLN concept, which implies that lymphatic metastasis initially occurs at SLN. In SLN navigation surgery, lymph node dissection beyond SLN can be omitted if SLN is metastasis free. Favorable outcomes are reported for malignant tumors such as breast cancer, malignant melanoma, and gastrointestinal cancer, and the application of this concept to other types of cancer is anticipated. In this study, the implications of the SLN concept on laryngeal cancer were investigated by measuring the feasibility of determining indications for neck dissection according to the presence of metastasis to SLN. Methods: Eleven patients suffering from laryngeal cancer staged T2 or higher and clinically N0 were enrolled in this study. Ninety-nine mTc-phytate (74MBq/mL) was injected submucosally by 0.2 mL into the several sites adjacent to the tumor using endoscope on the day preceding surgery. The lymphoscintigrams were performed 2 hours after injection. SLNs were surveyed using a gamma probe immediately prior to incision and during neck dissection. Histopathological examinations were performed on the resected lymph nodes. Results: One to three SLNs were confirmed by preoperative lymphoscintigraphy and intraoperatively by gamma probe in all of the cases. The pathological evaluation of SLNs revealed that 4 cases had subclinical metastasis, showing consistency with the sentinel node concept. In other 7 cases, metastasis to SLN as well as to other lymph nodes was negative, reflecting the correct lymph node status. No false-negative results were observed. Conclusion: Although more cases need to be studied in order to determine the feasibility of applying the SLN concept to laryngeal cancer, so far all cases were consistent with the sentinel node concept. Significance: SLN detection may be useful for determining indications for neck dissection in N0 laryngeal cancer. Support: None reported.

T-588 Prevents Motoneuron Loss after Recurrent Laryngeal Nerve Injury

Problem: Unsuccessful reinnervation or neurofunctional recovery after recurrent laryngeal nerve injury may attribute to the loss of motoneurons in nucleus ambiguus. To assess the possibility of new drug therapy for injury of these motoneurons, we examined the neuroprotective effect of a novel neuroprotective compound, T-588 on the motoneuron loss in nucleus ambiguus after adult rat vagal nerve avulsion. Methods: The left vagal nerves of 12-week-old Sprague-Dawley rats were avulsed and removed at the level of jugular foramen. After the operation, the animals were freely administrated water containing 0.05% T-588 (Toyama Chemical Co Ltd, Toyama, Japan) solution. Four weeks after the operation, the number of surviving motoneurons in nucleus ambiguus was counted after Nissl staining to evaluate the neuroprotective effect of T-588. Results: In T-588-treated animals, a significantly larger number of surviving motoneurons in nucleus ambiguus was observed compared to control animals 4 weeks after the operation. Conclusion: Oral administration of T-588 after vagal nerve avulsion improved the survival of motoneurons in nucleus ambiguus. Significance: Oral administration of T-588 may prevent the degeneration of motoneurons in nucleus ambiguus in adult humans with recurrent laryngeal nerve injury, and could be a new treatment modality for recurrent laryngeal nerve paralysis. Support: None reported.

Neurofunctional recovery after GDNF gene transfer in the recurrent laryngeal nerve-injured rat

Abstract Problem: To assess the possibility of gene therapy for recurrent laryngeal nerve (RLN) injury, we examined the neuroprotective effect after glial cell line-derived neurotrophic factor (GDNF) gene transfer into nucleus ambiguus in a rat vagal nerve avulsion model. Morphological preservation of motoneurons has previously been demonstrated (Brain Res 962: 61–67, 2003). However, functional recovery of RLN after gene therapy has not been demonstrated. In the present study, we examined neurofunctional recovery after GDNF gene transfer in a rat RLN injury model. Methods: The left RLN of Sprague-Dawley rats was crushed for 60 seconds with forceps. Immediately after the crush and confirmation of left fixed vocal fold, an adenoviral vector encoding beta-galactosidase gene (AxCALacZ) or GDNF (AxCAhGDNF) was directly injected into the crushed site of RLN. Transgene expression by AxCALacZ in nucleus ambiguus was examined by X-gal histochemistry. Measurement of conduction velocity (CV) of RLN and laryngoscopy to check motion recovery of the left vocal fold were performed 2 and 4 weeks postoperation. The cross-section of RLN was histologically examined after epon/toluidine blue staining. Results: Motoneurons and their neurites in nucleus ambiguus were labeled with X-gal staining 4 days after AxCALacZ injection, indicating successful induction of foreign gene into the nucleus ambiguus by retrogradely transported adenoviral vector from the crushed site. In comparison between AxCAhGDNF-injected, AxCALacZ-injected, and noninjected animals, significantly better CV of RLN and better vocal fold motion recovery rate were observed in AxCAhGDNF-injected animals 2 and 4 weeks postoperation. RLN cross section of AxCAhGDNF-injected animals showed better myelination compared to the controls. Conclusion: Injection of adenoviral vectors into the crushed site of RLN successfully induced the foreign gene into motoneurons in nucleus ambiguus. Adenoviral GDNF gene transfer after RLN crush improved the CV of RLN and motion recovery of the paralyzed vocal fold. Significance: Adenoviral GDNF gene therapy may provide neurofunctional recovery in humans with a paralyzed vocal fold. Support: None reported.