Description and Validation of a Simple Histological Nerve Tissue Scoring System
Authors: Hundepool C. A. , Bulstra L. F. , Shin D. M. , Bishop A. T. , Shin A. Y. 
Published: September 2016

Objective: The current gold standard treatment for peripheral nerve gaps remains the use of nerve autografts. Decellularized nerve allografts provide a potential ideal alternative, avoiding important disadvantages of the autograft such as donor site morbidity and the limited supply without the need for immunosuppressive therapy. In the ideal decellularized nerve allograft, all cellular debris is removed while maintaining the nerves’ ultrastructure. For the optimization of processing techniques for nerve allografts, it is essential to compare the effect of different processing techniques on a histological level. Although several studies describe some form of (subjective) histological scoring system, evaluation techniques are not uniform and nothing has been validated yet. A validated method that is simple and could easily be adopted by other groups would allow for a more standardized comparison of nerve decellularization techniques. Therefore, the aim of this study was to describe and validate an easy, fast, and inexpensive method to compare different nerve decellularization strategies on a histological level. Materials and Methods: Fifty rat sciatic nerve and 70 human nerve segments (35 sensory and 35 motor) were used for this study. Nerve segments were processed using 5 different processing techniques and compared with unprocessed nerve segments (control). Three different stainings were used to visualize general morphology and different components of the nerve tissue. Toluidine blue was used for visualization of myelin sheaths, hematoxylin-eosin (H&E) for general morphology, and laminin y-l for visualization of the basal lamina. All slides were scored by 4 independent observers who were blinded for the different processing techniques used. Each slide was given a score from 1 to 5 where 5 resembles a “perfect” structure similar to the native nerve and 1 resembles severe structural damage or absence of the component. To validate this scoring system, the agreement between the observers was calculated using the intraclass correlation coefficient (ICC). Results: The 4 different observers similarly scored the processed nerve segments. Significant differences between the processing groups could be identified. We found a good correlation between the different observers for the Toluidine Blue (ICC = .73, 95% confidence interval [CI], 0.66-0.79), H&E (ICC = .72, 95% CI, 0.64-0.78), and Laminin staining (ICC = .74, 95% CI, 0.67-0.80). Conclusion: This study demonstrates the validity of the scoring system to evaluate nerve tissue after different processing protocols. A good correlation was found for the scoring of the structural integrity (Toluidine Blue, H&E, and Laminin). This method is suitable to compare different processing techniques in vitro on the route to engineering the ideal nerve allograft. Use of this method by other groups will make it possible to compare results of different nerve processing studies. Using valid outcome measurements is essential when striving to create new innovative options for nerve reconstruction.