Rapid and Precise Semi-Automatic Axon Quantification in Human Peripheral Nerves

Abstract

We developed a time-efficient semi-automated axon quantification method using freeware in human cranial nerve sections stained with paraphenylenediamine (PPD). It was used to analyze a total of 1238 facial and masseteric nerve biopsies. The technique was validated by comparing manual and semiautomated quantification of 129 (10.4%) randomly selected biopsies. The software-based method demonstrated a sensitivity of 94% and a specificity of 87%. Semi-automatic axon counting was significantly faster (p < 0.001) than manual counting. It took 1 hour and 47 minutes for all 129 biopsies (averaging 50 sec per biopsy, 0.04 seconds per axon). The counting process is automatic and does not need to be supervised. Manual counting took 21 hours and 6 minutes in total (average 9 minutes and 49 seconds per biopsy, 0.52 seconds per axon). Our method showed a linear correlation to the manual counts (R = 0.944 Spearman rho). Attempts have been made by several research groups to automate axonal load quantification. These methods often require specific hard-and software and are therefore only accessible to a few specialized laboratories. Our semi-automated axon quantification is precise, reliable and time-sparing using publicly available software and should be useful for an effective axon quantification in various human peripheral nerves. Microscopic analysis of peripheral nerves is key for many clinical and research based projects. Peripheral nerves have been analyzed through multiple methods, which can generally be categorized into 'manual' , 'automated' and 'semi-automated' methods. Here, the terms for 'manual' and 'fully automated' morphometry will be used as previously described 1-4. 'Semi-automated' will be used synonymously with Urso-Baiardas 'interactive automated' approach; an automated method with the opportunity for manual preparation or alteration 3. In the past, no prime and uniform method could be found, that is simple, cost efficient and time sparing. Therefore, tendentially, small research collectives use manual methods for analysis 1,2,5. Attempts have been made by several research groups throughout medical and scientific research, to automate this process 3,6-8. Unfortunately, it is often found, that these methods are either highly specialized, thus accessible to only few expert laboratories, or, in the case of highly developed software and hardware, very costly 3,9. By example, Marina et al. have coined a method which, similar to this project, focuses on simpler semi-automated anaylsis 10. Other research groups such as Hunter et al. focus on highly specialized methods, which are able to produce a wide range of data and process numerous variables 11. The semi-automated quantification method proposed in this study was developed as part of a greater study on Human facial nerves, for which a time sparing, cost efficient and user-friendly method of axonal quantification was required. Patients with facial palsy, caused by dysfunction of the seventh cranial nerve, suffer emotional distress and are often socially isolated 1...

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“…Another technical observation is the possibility to manually select the brightness threshold during FIAC, which is also carried out in similar studies ( 13 , 24 ). The most common automatization problem is distinguishing artifactual features from actual nerve fibers ( 25 ). Therefore, for a meaningful quantification of the fluorescent structures, the operator can select an appropriate threshold to avoid false positives or artifacts and ensure accuracy through operator validation.…”

Validation of the Use of Automated and Manual Quantitative Analysis of Corneal Nerve Plexus Following Refractive Surgery

“…However, manual marking is time-consuming and labour-intensive with an analysis time of approximately 20 min per image. Given its laborious nature, manual tracing is subject to user fatigue, increasing the possibility of unreliable tracing, and lowering productivity due to the limitation in the number of images analysed per day [ 23 ]. In addition, it is highly user-dependent and requires the expertise of the observer in both identifying and tracing the tortuous nerve contours.…”

“…In addition, it is highly user-dependent and requires the expertise of the observer in both identifying and tracing the tortuous nerve contours. Thus, the subjective nature of nerve identification is more obvious in manual quantification, resulting in inconsistencies and a need for both inter-observer and intra-observer analysis [ 23 ]. This is illustrated by the worse inter-observer agreement compared to the intra-observer agreement.…”

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“…Of note, the MMB is commonly represented by one or two branches [ 25 ]. The microanatomy of the MMB has been studied by our group previously, demonstrating its axonal load, fascicle structure, and diameter [ 18 , 26 ]. With its function of lowering the corner of the mouth, the DAO is crucial for expressing sorrow and anger, for example [ 27 ].…”