Allotransplanted Neurons Used to Repair Peripheral Nerve Injury Do Not Elicit Overt Immunogenicity

A major problem hindering the development of autograft alternatives for repairing peripheral nerve injuries is immunogenicity. We have previously shown successful regeneration in transected rat sciatic nerves using conduits filled with allogeneic dorsal root ganglion (DRG) cells without any immunosuppression. In this study, we re-examined the immunogenicity of our DRG neuron implanted conduits as a potential strategy to overcome transplant rejection. A biodegradable NeuraGen® tube was infused with pure DRG neurons or Schwann cells cultured from a rat strain differing from the host rats and used to repair 8 mm gaps in the sciatic nerve. We observed enhanced regeneration with allogeneic cells compared to empty conduits 16 weeks post-surgery, but morphological analyses suggest recovery comparable to the healthy nerves was not achieved. The degree of regeneration was indistinguishable between DRG and Schwann cell allografts although immunogenicity assessments revealed substantially increased presence of Interferon gamma (IFN-γ) in Schwann cell allografts compared to the DRG allografts by two weeks post-surgery. Macrophage infiltration of the regenerated nerve graft in the DRG group 16 weeks post-surgery was below the level of the empty conduit (0.56 fold change from NG; p<0.05) while the Schwann cell group revealed significantly higher counts (1.29 fold change from NG; p<0.001). Major histocompatibility complex I (MHC I) molecules were present in significantly increased levels in the DRG and Schwann cell allograft groups compared to the hollow NG conduit and the Sham healthy nerve. Our results confirmed previous studies that have reported Schwann cells as being immunogenic, likely due to MHC I expression. Nerve gap injuries are difficult to repair; our data suggest that DRG neurons are superior medium to implant inside conduit tubes due to reduced immunogenicity and represent a potential treatment strategy that could be preferable to the current gold standard of autologous nerve transplant

The feasibility analysis of omission of elective irradiation to level IB lymph nodes in low-risk nasopharyngeal carcinoma based on the 2013 updated consensus guideline for neck nodal levels

Abstract Background Level IB metastasis is rare in nasopharyngeal carcinoma (NPC). The purpose of this study is to investigate the high-risk factors for level IB metastasis and evaluate the feasibility of omission of elective irradiation to level IB in the low-risk subgroups in NPC. Methods This retrospective study identified 532 patients with NPC treated by definitive radiation in our institution from 2009 to 2010. Level IB nodes were electively irradiated based on the physician’s decision. Diagnostic head and neck MRIs were reviewed. The involvements of nodal levels were evaluated according to 2013 updated guidelines of RTOG. The correlations of level IB metastasis and other factors were studied using Chi-square test and logistic regression model. Log-rank tests were used to compare survival rates. Cox proportional-hazards models were used to evaluate the effect of various factors. Patient-reported xerostomia was recoded in every follow-up and the extents of delayed xerostomia at 1 year post-radiation were compared between those with/without elective level IB irradiation. Results N stage, bilateral nodal metastasis, level II involvement, level IIA involvement, level IIA with multiple levels involvement, maximal axial diameter (MAD) of level IIA nodes > 20 mm, MAD of neck lymph nodes > 30 mm, necrosis of level IIA nodes, extracapsular spread of level IIA correlated with level IB metastasis by univariate analysis. In multivariate analysis (MVA), bilateral nodal involvement, MAD of level IIA nodes > 20 mm or extracapsular spread of level IIA nodes, were independent predictive factors for level IB metastasis. Patients without either these factors were denoted low-risk group and the rest high-risk group. Of the low-risk group, there was no significant difference of regional control and overall survival (OS) between those with or without elective irradiation. The percentage of level IB recurrence of those without elective irradiation was 0.46%. Elective level IB irradiation was not significant upon MVA both for regional control and OS. Of the high-risk group, elective level IB irradiation was marginal significant for regional control, but not for OS upon MVA. No regional recurrence located at level IB. Overall, omission of elective irradiation to level IB reduced the mean doses of submandibular glands, but did not improve patient-reported xerostomia. Conclusion For patients without high-risk factors of level IB metastasis, omission of elective level IB irradiation did not impair regional control and OS in NPC

Development and validation of a model for temporal lobe necrosis for nasopharyngeal carcinoma patients with intensity modulated radiation therapy

Abstract Purpose To develop and validate a quantitative complication model of temporal lobe necrosis (TLN). To analyze the effect of clinical and dosimetric factors on TLN. Patients and methods In this study the prediction model was developed in a training cohort that consisted of 256 nasopharyngeal carcinoma (NPC) patients from January 2009 to December 2009. Dosimetric and clinical factors were extracted for model building. Dosimetric factors including the maximum dose, minimum dose, mean dose, dose covering specific volume and dose of percentage volume. Clinical factors include age, gender, T/N-stage, overall stage, diabetes and hypertension. LASSO (least absolute shrinkage and selection operator) regression model was used for feature selection, and prediction model building. A testing cohort containing 493 consecutive patients from January 2010 to December 2010 was used for model validation. The performance of the prediction model was assessed with respect to its calibration, discrimination. Results The prediction model, which consisted of two dosimetric features (D0.5cc and D10), is significantly associated with LN status (P < .001 for both training and testing cohorts). None of clinical factors show direct prediction value. The model shows good discrimination, with a C-index of 0.685 (95% CI: 0.6048–0.765) on testing set, and a consistent trend in calibration on testing set. Conclusion This study presents a prediction model can be conveniently used to facilitate the individualized prediction of TLN in patients with NPC. Clinical factors have no direct impact on TLN

Additional file 1: of The feasibility analysis of omission of elective irradiation to level IB lymph nodes in low-risk nasopharyngeal carcinoma based on the 2013 updated consensus guideline for neck nodal levels

Table S1. Comparison of clinical factors between high-risk and low-risk subgroups. Table S2. Distribution of regional recurrence by risk-stratified subgroups. Table S3. The grades of xerostomia by elective irradiation to level IB. Figure S1. Isodose distribution of submandibular glands. (PDF 237 kb

Histological analysis of the sciatic nerve 16 weeks post-PNI.

<p>For anatomical terminology, refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031675#pone-0031675-g001" target="_blank"><i>Fig. 1E</i></a>. (A) Myelination (MBP, green) of the regenerated axons (MAP2, red). Inset show the structure of individual myelinated axon fascicles, enlarged from 40× image. Axial sections 40×, longitudinal sections 20×. Thickness = 15 um. (B) Expression of laminin (green) was consistent with results from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031675#pone-0031675-g003" target="_blank"><i>Fig. 3A</i></a>. DAPI (blue), 20×. Thickness = 15 um.</p

Evaluating the health of cultured neurons.

<p>(A) At the end of antimitotic treatment (day 3 and 4), the DRG cells (MAP-2 immunopositive neurons; green) had extended axons, and non-neuronal cells were essentially nonexistent. (B) Schwann cells demonstrated strong GFAP (red) expression and an elongated shape and dipolar morphology at day 2 and 3. 20×, Scale bar: 50 um. DAPI (blue).</p

Surgical Procedures.

<p>(A) After skin incision, an 8 mm length of sciatic nerve was excised. (B) A NeuraGen® nerve guide tube of 2.0 mm ID×1.0 cm length was used to provide a physical guide for axons sprouting from the proximal nerve stump to reach the disconnected nerve segment. (C) The rats were sacrificed at 16 weeks post-PNI and sciatic nerves were harvested by excising 1.0 cm outside the NG tube. (D) The tube (top) is distinguishable from the contralateral healthy tissue (bottom) as an enlarged section of the nerve. The contralateral nerve served as a control for each rat. (E) For histological analysis, frozen sections were cut and examined at five locations: nerve proximal (P) and distal (D) to the tube and proximal (Lp), distal (Ld), and middle (Lm) longitudinal regions inside the tube.</p

Histological and quantitative analyses of macrophage presence.

<p>For anatomical terminology, refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031675#pone-0031675-g001" target="_blank"><i>Fig. 1E</i></a>. (A) ED1 IHC shows macrophage (red) location and number in each group in neuronal structure (NF-200, green). Note Sham, Wistar-DRG, NG, and Wistar Schwann represent macrophage presence in increasing order in all tissue sections. DAPI (blue), 20×. Thickness = 15 um. (B) Quantification and statistical analysis of ED1 immunoreactive cells. Macrophage count reflects immunohistochemistry microscopy analysis in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031675#pone-0031675-g004" target="_blank"><i>Fig. 4A</i></a>. ANOVA Bonferroni and Tukey multiple comparison post tests determined significant differences in the mean macrophage count in each group: *P<0.05; **P<0.01; ***P<0.001. Both post tests found the same results. Error bars represent ± standard error of the mean.</p

MHC I immunoreactivity (red) in neuronal structure (NF-200, green).

<p>For anatomical terminology, refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031675#pone-0031675-g001" target="_blank"><i>Fig. 1E</i></a>. (A) Increased MHC I immunoreactivity were observed in NG, DRG, and Schwann groups compared to the Sham group. DAPI (blue), 20×. Thickness = 15 um. (B) Axon regeneration estimated by NF-200 expressing axons. Axon density in distal stump was calculated and expressed as standard optical density (SOD) for each group. n = 3, 40× image of 10 um sections.</p